Multi-transgenic pig for xenotransplantation

Abstract

To provide a donor animal which can provide a pulmonary xenograft having an improved functionality.SOLUTION: The present invention is directed to transgenic animals (e.g., transgenic porcine animals) comprising multiple genetic modifications that advantageously render these animals suitable donors for xenotransplantation. The present invention extends to organs, organ fragments, tissues and cells derived from these animals and their therapeutic use. The present invention further extends to methods of making such animals. In the first aspect, the present invention provides the transgenic porcine animals that include at least four transducing genes that are incorporated and expressed in the single gene locus under the control of at least two promoters and lack expression of alpha-1,3-galactosyltransferase.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 216,225, filed on September 9, 2015, and U.S. Provisional Patent Application No. 62 / 256,068, filed on November 16, 2015, the contents of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Because pigs share many anatomical and physiological characteristics with humans, pigs have been the focus of most xenotransplantation research. Pigs also have a relatively short gestation period, can be raised in a sterile environment, and do not raise the same ethical issues associated with animals commonly used as food sources (e.g., primates). Scientific knowledge and expertise in the field of xenotransplantation from pigs to primates have grown rapidly over the past decade, resulting in the fairly long-term survival of primate recipients of life-saving pig xenografts. (Cozzi et al., Xenotransplantation, Vol. 16: 203-214, 2009). Recently, significant achievements have been reported in the field of organ xenotransplantation. (Ekser et al., 2009, Transplant Immunology Jun, Vol. 21(2): 87-92).

[0003] Considerable progress has been made in overcoming the biological barriers to the use of pig organs in preclinical models, and organ function and recipient survival have reached from several months to several years in some cardio-renal systems (Mohiuddin MM et al., Am J Transplant, 2014; Vol. 14: 488-489; Iwase H et al., Xenotransplantation, 2015; Vol. 22: 302-309 Page. Higginbotham L et al., Xenotransplantation, 2015; 22: 221-230). However, progress to date has been substantial with respect to the heart and kidney, but has not yet reached the level of being transferred to humans. Furthermore, other organs such as the lungs pose even greater challenges. For example, survival of life-supporting lung xenografts is limited to a few days in primates (Laird et al., June 2016, www.cotransplantation.com, 21(3)).

[0004] Lung transplantation is a procedure approved for advanced lung diseases. First performed in 1963, more than 32,000 lung transplants have been carried out worldwide since then. The majority of procedures are cadaveric transplants, and the donor lungs are obtained from patients who are brain dead but still in a life-support state. The limitation in the number of cadaveric donor lungs led to the development of living donor lobar lung transplantation (LDLLT) in the 1990s, where two or more living patients donate segments (lobes) of the lung. However, the donor pool remains relatively scarce, and the long-term outcomes of transplantation remain hampered by immunosuppressive regimens.

[0005] Xenotransplantation (transplantation of organs, tissues, and cells from donors of different species) may effectively address the shortage of human donor lungs. Conveniently, xenografts are (i) predictably and non-urgently supplied; (ii) produced in a controlled environment; and (iii) available for pre-transplant characterization and research. However, compared to other organs, the unique anatomical structure of the lungs, with a large surface area of alveolar epithelium closely associated with vascular endothelium, as well as a robust immune surveillance and rapid response system, is predisposed to inducing inflammation in advance and is extremely susceptible to the resulting effects (den Hengst WA et al., Am J Physiol Heart Circ Physiol, 2010; 299: H1283-H1289; Ranieri VM et al., JAMA, 1999; 282: 54-61).

[0006] Although advantageous in many respects, xenotransplantation creates a more complex immunological scenario than allotransplantation. The most significant barriers to xenotransplantation from pigs to primates are the rejection of the transplanted organ by a cascade of immune mechanisms that can be divided into three phases: hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), and T cell-mediated cellular rejection. HAR is a very rapid event that results in irreversible graft damage and loss within minutes to hours after graft reperfusion.

[0007] Considerable efforts have been directed at addressing the immunological barriers posed by xenotransplantation through genetic modification of donor animals. Genetically modified pigs lacking the alpha-1,3-Gal epitope (the major xenoantigen that induces HAR of pig-to-primate xenografts) are thought to be a basis for further genetic modification to address other rejection mechanisms and incompatibilities between porcine and primate blood coagulation systems. Although multiple genetic modifications will probably be required for the success of xenotransplantation, they pose problems, including production-related issues. It is clear that the generation of transgenic pigs stably expressing multiple immunomodulatory transgenes is essential for overcoming xenograft rejection.

[0008] The generation of multi-transgenic pigs by traditional mating of pigs containing a single transgene has been used and has had much success to date (Ekser et al., 2009, Transplant Immunology Jun;21(2):87-92; Laird et al., June 2016, www.cotransplantation.com, Vol. 21, No. 3). However, mating is time-consuming, expensive, and the consistent expression levels of the transgenes may eventually become a problem.

[0009] Recently, the use of polycistronic expression systems has been developed for inserting multiple transgenes into various cell types and animals. The feasibility of using these systems to generate multi-transgenic pigs has been suggested.

[0010] Deng et al. (PLOS ONE, www.plosone.org, May 2011, 6(5), e19986) produced transgenic pigs expressing four fluorescent proteins using a 2A peptide bicistronic system and nuclear transfer with random integration of the transgene.

[0011] Jeong et al. (PLOS ONE, www.plosone.org, May 2013, 8(5), e63241 page) reported the production of transgenic pigs expressing complement regulatory factor CD59 and H-transferase gene using an IRES-mediated tricistronic vector system and nuclear transfer. Jeong et al. actually attempted to express three genes using this tricistronic system, but the third gene, CD55, was not expressed in pigs despite its presence in the IRES vector.

[0012] Hurh et al. (PLOS ONE, www.plosone.org, July 2013, 8(7), e70486) generated transgenic pig fibroblasts using a bicistronic T2A expression system and analyzed the expression of transgenic proteins using this system. They reported that efficient expression of the downstream gene can be achieved if the expression of the upstream gene is efficient.

[0013] Multitransgenic pigs using a polycistronic expression system that results in stable and sufficient integration and expression of the transgene have not yet been produced. Therefore, it has not yet been proven whether this strategy can be a viable alternative to the traditional mating approach commonly used to generate multitransgenic pigs.

[0014] There is still a need for improved donor animals for xenotransplantation therapy. In particular, there is still a need for donor animals that can provide lung xenografts with improved functionality.

Prior Art Documents

Non-Patent Literature

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Summary of the Invention

Means for Solving the Problems

[0016] The present invention is directed to transgenic animals (e.g., transgenic pig animals) that comprise a plurality of genetic modifications that advantageously render those animals suitable donors for xenotransplantation. The present invention extends to organs, organ fragments, tissues and cells derived from these animals, as well as their therapeutic uses. The present invention further extends to methods of producing such animals.

[0017] In a first aspect, the present invention provides a transgenic pig comprising at least four transgenes integrated and expressed at a single locus under the control of at least two promoters, the transgenic pig lacking expression of alpha1,3-galactosyltransferase.

[0018] The single locus can be any suitable locus. In one embodiment, the single locus is an unmodified natural locus. In an alternative embodiment, the single locus is a modified natural locus. The locus can be modified by any suitable means including, but not limited to, insertions, deletions, or substitutions mediated by gene editing tools. In certain embodiments, the modified natural locus comprises transgenic DNA. The transgenic DNA can be, for example, a selectable marker gene. In other embodiments, the transgenic DNA is a landing pad as further described herein.

[0019] In certain embodiments, the single locus is AAVS1, ROSA26, CMAH, β4GalNT2 or GGTA1. According to this embodiment, the locus may be natural or modified.

[0020] In an exemplary embodiment, a single locus is a native GGTA1 or a modified native GGTA1. In certain embodiments, the modified native GGTA1 locus comprises a selectable marker gene, such as neo. In other embodiments, the modified native GGTA1 locus comprises an insertion, deletion or substitution mediated by a gene editing tool. In still other embodiments, the modified native GGTA1 locus comprises a landing pad for facilitating gene targeting.

[0021] The promoters may be different. In an exemplary embodiment, the promoters are endogenous, exogenous or a combination thereof. In an exemplary embodiment, the promoters are constitutive, regulatable or a combination thereof. In certain embodiments, at least one of the promoters is regulatable (e.g., a tissue-specific or inducible promoter).

[0022] In an exemplary embodiment, the transgenic pig comprises four transgenes, where the four transgenes are expressed as a first and a second polycistron, a first promoter controls the expression of the first polycistron, and a second promoter controls the expression of the second polycistron.

[0023] In an exemplary embodiment, the transgenic pig comprises four transgenes, and each of at least four transgenes is controlled by a dedicated promoter.

[0024] In certain embodiments, the transgenic pig comprises at least four transgenes, where the at least four transgenes are integrated and expressed at a single locus under the control of at least two promoters, at least one of the promoters is constitutive (e.g., CAM), at least one of the promoters is tissue-specific (e.g., an endothelium-specific promoter such as ICAM-2), and the pig lacks the expression of alpha1,3-galactosyltransferase.

[0025] In another specific embodiment, the transgenic pig contains at least four transgenes, where the at least four transgenes are integrated and expressed at a single locus under the control of at least two promoters, at least two of the promoters are constitutive, and the pig lacks the expression of alpha 1,3 galactosyltransferase.

[0026] The transgenes may be different. In an exemplary embodiment, the transgenes are anticoagulant factors, complement inhibitors, immunomodulatory factors, cytoprotective transgenes, or combinations thereof.

[0027] In certain embodiments, at least one of the transgenes is an anticoagulant factor. In one embodiment, the anticoagulant factor is TBM, TFPI, EPCR, or CD39. In certain embodiments, at least two of the transgenes are anticoagulant factors.

[0028] In certain embodiments, at least one of the transgenes is a complement regulatory factor, such as a complement inhibitor. In one embodiment, the complement inhibitor is CD46, CD55, or CD59.

[0029] In certain embodiments, at least one of the transgenes is an immunomodulatory factor. The immunomodulatory factor may be, for example, an immunosuppressive factor. In one embodiment, the immunosuppressive factor is porcine CTLA4-IG or CIITA-DN. In certain embodiments, at least one of the transgenes is CD47.

[0030] In an exemplary embodiment, the transgenic animal includes genetic modifications in addition to the expression of at least one additional, i.e., multiple, transgenes and the lack of expression of alpha Gal.

[0031] Further genetic modifications may vary. In an exemplary embodiment, at least one genetic modification is a gene knockout, gene knock-in, gene replacement, point mutation, deletion, insertion or substitution of a gene, gene fragment or nucleotide, large genomic insertion, or a combination thereof.

[0032] In certain embodiments, a single locus is not GGTA1 and at least one additional genetic modification comprises a knockout of the alpha 1,3 galactosyltransferase gene.

[0033] In other embodiments, the additional genetic modification comprises the incorporation and expression of at least one additional transgene. In one embodiment, the additional transgene is the human CD46 gene, human HLA - 3 and / or humanized vWF or chimeric porcine - human vWF gene.

[0034] In certain embodiments, at least one additional genetic modification is a modification of the porcine vWF locus to reduce or eliminate spontaneous aggregation of human platelets.

[0035] In certain embodiments, at least one additional genetic modification is a knockout of a porcine gene. The porcine gene may be, in certain embodiments, β4GalNT2, CMAH, isogloboside 3 synthase, Forssman synthase or vWF.

[0036] In certain embodiments, at least one additional genetic modification comprises the incorporation and expression of at least two or more additional transgenes. In one embodiment, two or more additional transgenes are incorporated and expressed at a single second locus.

[0037] In an exemplary embodiment, the transgenic pig contains at least six transgenes, wherein (i) at least four transgenes are integrated and expressed at a first single locus (e.g., GGTA1) under the control of at least two promoters, and (ii) at least two transgenes are integrated and expressed at a second single locus (e.g., β4GalNT2 or CMAH) under the control of at least one promoter, and the pig lacks the expression of alpha 1,3 galactosyltransferase.

[0038] In a second aspect, the invention is an organ or organ fragment derived from the transgenic pig of the first aspect of the invention.

[0039] In an exemplary embodiment, the organ is a lung, liver, heart or pancreas.

[0040] In an exemplary embodiment, the organ fragment is a lung fragment, liver fragment, heart fragment or pancreas fragment.

[0041] In a third aspect, the invention is a tissue derived from the transgenic pig of the first aspect of the invention.

[0042] In an exemplary embodiment, the tissue is epithelial tissue or connective tissue.

[0043] In a fourth aspect, the invention is a cell derived from the transgenic pig disclosed herein.

[0044] In an exemplary embodiment, the cell is an islet cell.

[0045] In a fifth aspect, the present invention is a method of producing a transgenic pig that expresses at least four transgenic genes but lacks the expression of alpha 1,3 galactosyltransferase, the method comprising: (i) integrating at least four transgenes under the control of at least two promoters at a single locus in the pig genome to provide a polygenic pig genome; (ii) enabling cells containing the polygenic pig genome to mature into a transgenic pig.

[0046] In an exemplary embodiment, the pig genome is a somatic pig genome, the cells are pig zygotes, and the pig zygotes are provided by somatic cell nuclear transfer (SCNT) and further transferring the polygenic pig genome into the reconstructed SCNT zygotes by microinjection. Optionally, the somatic genome and / or the polygenic pig genome can contain one or more additional genetic modifications. In one embodiment, at least one genetic modification is selected from gene knockout, gene knock-in, gene replacement, point mutation, deletion, insertion or substitution of a gene, gene fragment or nucleotide, large genomic insertion or a combination thereof.

[0047] In an exemplary embodiment, the pig genome is selected from the group consisting of a germline pig genome, a zygotic pig genome, an embryonic pig genome or a blastocyst pig genome. Optionally, the pig genome or the polygenic pig genome contains at least one additional genetic modification. In one embodiment, at least one genetic modification is selected from gene knockout, gene knock-in, gene replacement, point mutation, deletion, insertion or substitution of a gene, gene fragment or nucleotide, large genomic insertion or a combination thereof.

[0048] The method of incorporation may vary. In an exemplary embodiment, the incorporation includes biological transfection, chemical transfection, physical transfection, virus-mediated transduction or transformation, or combinations thereof. In a particular embodiment, the incorporation includes cytoplasmic microinjection. In another particular embodiment, the incorporation includes pronuclear microinjection.

[0049] Consistent with the first aspect of the present invention, the single locus may vary.

[0050] In an exemplary embodiment, the single locus contains transgenic DNA. In a particular embodiment, the transgenic DNA is a landing pad containing one or more recognition sites for at least one polynucleotide modifying enzyme. The polynucleotide modifying enzyme may vary. In certain particular embodiments, the polynucleotide modifying enzyme is an engineered endonuclease, a site-specific recombinase, an integrase, or combinations thereof.

[0051] In one embodiment, the engineered endonuclease is a zinc finger nuclease, a transcription activator-like effector nuclease, or a clustered regularly interspaced short palindromic repeats / Cas9 nuclease.

[0052] In one embodiment, the site-specific recombinase is lambda integrase, Cre recombinase, FLP recombinase, gamma-delta resolvase, Tn3 resolvase, ΦC31 integrase, Bxb1-integrase, R4 integrase, or combinations thereof.

[0053] In one embodiment, the single locus is a natural or modified locus selected from GGTA1, CMAH, β4GalNT2, the AAVS1 locus, and Rosa26.

[0054] In embodiments where the single locus is not the GGTA1 locus, further genetic modifications include knocking out the alpha1,3-galactosyltransferase gene. Other knockouts contemplated by the present invention as further genetic modifications include knockouts of the porcine β4GalNT2 gene, the CMAH gene, the β4GalNT2 gene, vWF, or combinations thereof.

[0055] In exemplary embodiments, at least one further genetic modification includes the incorporation and expression of at least one further transgene. In certain embodiments, the transgene is human CD46, human HLA-E, humanized vWF, chimeric porcine-human vWF, or fully human vWF.

[0056] In a sixth aspect, the present invention is a transgenic pig or production herd produced by the method of the fifth aspect of the present invention.

[0057] In a seventh aspect, the present invention is a method of mating a transgenic pig of the present invention with a second transgenic pig, wherein the second transgenic pig is characterized by one or more genetic modifications.

[0058] In exemplary embodiments, the second transgenic pig is characterized by one or more genetic modifications such as gene knockout, gene knock-in, gene replacement, point mutation, deletion, insertion or substitution of a gene, gene fragment or nucleotide, large genomic insertion, or combinations thereof.

[0059] In an eighth aspect, the present invention is a transgenic pig or production herd produced by the method of the seventh aspect of the present invention.

[0060] In a ninth aspect, the present invention provides a method for treating a subject in need thereof by transplanting into the subject at least one organ, organ fragment, tissue or cell derived from a transgenic pig of the present invention.

[0061] In an exemplary embodiment, the organ or organ fragment is a lung or lung fragment, a kidney or kidney fragment, a liver or liver fragment, a pancreas or pancreas fragment, or a combination thereof.

[0062] In certain embodiments, the organ is a lung. In another particular embodiment, the organ fragment is a lung fragment. In an exemplary embodiment, the lung or lung fragment is transplanted in a subject having a progressive lung disease.

[0063] In an exemplary embodiment, the lung or lung fragment is transplanted in a subject having a progressive lung disease associated with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), alpha1-antitrypsin disease, or primary pulmonary hypertension.

[0064] In certain embodiments, the method includes administering to the subject one or more additional therapeutic agents. The one or more therapeutic agents may be different. In one embodiment, the therapeutic agents are antirejection agents, anti-inflammatory agents, immunosuppressive agents, immunomodulatory agents, antibacterial agents, and antiviral agents, and combinations thereof.

[0065] In a tenth aspect, the invention provides a transgenic pig having a genetic modification at the porcine vWF locus and lacking expression of alpha1,3-galactosyltransferase. The transgenic pig can include one or more additional genetic modifications.

[0066] In an exemplary embodiment, the transgenic pig has a genetic modification at the porcine vWF locus, incorporates and expresses at least four transgenes, and lacks expression of alpha1,3-galactosyltransferase. Examples of embodiments of the invention include the following items. (Item 1) A transgenic pig containing at least four transgenes, wherein the at least four transgenes are integrated and expressed at a single locus under the control of at least two promoters, and the pig lacks the expression of alpha 1,3 galactosyltransferase. (Item 2) The transgenic pig according to item 1, wherein the single locus is a natural locus. (Item 3) The transgenic pig according to item 1, wherein the single locus is a modified natural locus. (Item 4) The transgenic pig according to item 3, wherein the modified natural locus includes gene editing-mediated insertion, deletion or substitution. (Item 5) The transgenic pig according to item 3, wherein the modified natural locus contains transgenic DNA. (Item 6) The transgenic pig according to item 5, wherein the transgenic DNA contains a selectable marker gene. (Item 7) The transgenic pig according to item 5, wherein the transgenic DNA contains a landing pad. (Item 8) The transgenic pig according to item 1, wherein the single locus is selected from the group consisting of AAVS1, ROSA26, CMAH, β4GalNT2 and GGTA1. (Item 9) The transgenic pig according to item 1, wherein the single locus is the natural GGTA1 locus. (Item 10) The transgenic pig according to item 1, wherein the single locus is a modified GGTA1 locus. (Item 11) The transgenic pig according to item 1, wherein the single locus is a transgenic GGTA1 locus. (Item 12) The transgenic pig according to item 1, wherein at least one of the promoters is a constitutive promoter. (Item 13) The transgenic pig according to item 1, wherein at least one of the promoters is a regulatable promoter. (Item 14) The transgenic pig according to item 13, wherein the regulatable promoter is a tissue-specific promoter or an inducible promoter. (Item 15) The transgenic pig according to item 1, wherein at least one of the promoters is an exogenous promoter. (Item 16) The transgenic pig according to item 1, wherein the at least four transgenes are expressed as a first polycistron and a second polycistron, and the at least two promoters include a first promoter that controls the expression of the first polycistron and a second promoter that controls the expression of the second polycistron. (Item 17) The transgenic pig according to item 1, including at least four promoters, wherein each of the at least four transgenes is controlled by a dedicated promoter. (Item 18) The transgenic pig according to item 16, wherein the first promoter is a constitutive promoter and the second promoter is a tissue-specific promoter. (Item 19) The transgenic pig according to item 18, wherein the tissue-specific promoter is an endothelial cell-specific promoter. (Item 20) The transgenic pig according to item 16, wherein the first promoter and the second promoter are constitutive promoters. (Item 21) The transgenic pig according to item 1, wherein the at least two promoters include CAG and ICAM-2. (Item 22) The transgenic pig according to item 1, wherein the at least four transgenes are selected from the group consisting of anticoagulation factors, complement inhibitors, immunomodulatory factors, cytoprotective transgenes, and combinations thereof. (Item 23) The transgenic pig according to item 22, wherein the anticoagulation factor is selected from the group consisting of TBM, TFPI, EPCR, CD39, and combinations thereof. (Item 24) The transgenic pig according to item 22, wherein the complement inhibitor is selected from the group consisting of CD46, CD55, CD59, and combinations thereof. (Item 25) The transgenic pig according to item 22, wherein the immunomodulatory factor is an immunosuppressive factor. (Item 26) The transgenic pig according to item 25, wherein the immunosuppressive factor is selected from the group consisting of porcine CLTA4-IG, CIITA-DN, and combinations thereof. (Item 27) The transgenic pig according to item 22, wherein the immunomodulatory factor is CD47. (Item 28) The transgenic pig according to item 22, wherein the cytoprotective transgene is selected from the group consisting of HO-1, A20, and combinations thereof. (Item 29) The transgenic pig according to item 1, wherein at least two of the transgenes are anticoagulation factors. (Item 30) The transgenic pig according to item 29, wherein at least one of the transgenes is a cytoprotective transgene. (Item 31) The transgenic pig according to item 30, wherein at least one of the transgenes is an immunomodulatory factor. (Item 32) The transgenic pig according to item 31, wherein at least one of the transgenes is a complement inhibitor. (Item 33) The transgenic pig according to item 1, further comprising at least one additional genetic modification. (Item 34) The transgenic pig according to item 33, wherein the at least one additional genetic modification is selected from the group consisting of gene knockout; gene knock-in; gene replacement; point mutation; deletion, insertion or substitution of a gene, gene fragment or nucleotide; large genomic insertion; or a combination thereof. (Item 35) The transgenic pig according to item 33, wherein the single locus is not GGTA1 and the at least one additional genetic modification comprises knockout of the alpha1,3-galactosyltransferase gene. (Item 36) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises incorporation and expression of human CD46. (Item 37) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises modification of the porcine vWF locus to reduce or eliminate spontaneous aggregation of human platelets. (Item 38) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises incorporation and expression of chimeric porcine-human vWF. (Item 39) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises targeted inactivation of the porcine vWF gene and incorporation and expression of a fragment of the human vWF gene. (Item 40) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises incorporation and expression of human HLA-E. (Item 41) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises knockout of the β4GalNT2 gene. (Item 42) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises knockout of the CMAH gene. (Item 43) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises the integration and expression of at least two additional transgenes. (Item 44) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises the integration and expression of at least two additional transgenes at a second single locus. (Item 45) The transgenic pig according to item 33, wherein the at least one additional genetic modification comprises the integration and expression of at least four additional transgenes at a second single locus. (Item 46) The transgenic pig according to item 44, wherein the single locus is GGTA1 and the second single locus is β4GalNT2. (Item 47) The transgenic pig according to item 44, wherein the single locus is GGTA1 and the second single locus is CMAH. (Item 48) The transgenic pig according to item 33, wherein the at least one additional genetic modification results in the elimination or reduction of the expression of at least one native gene. (Item 49) The transgenic pig according to item 48, wherein the at least one native gene is selected from the group consisting of CMP-NeuAc hydroxylase, isogloboside 3 synthase, β4GalNT2, vWF, Forssman synthase, or combinations thereof. (Item 50) Organs derived from the transgenic pigs according to items 1 to 49. (Item 51) Lungs or lung fragments derived from the transgenic pigs according to items 1 to 49. (Item 52) Tissues derived from the transgenic pigs according to items 1 to 49. (Item 53) Cells derived from the transgenic pigs described in Items 1 to 49. (Item 54) A method for producing a transgenic pig that expresses at least four transgenic genes but lacks the expression of alpha1,3-galactosyltransferase, the method comprising: (i) integrating at least four transgenes under the control of at least two promoters into a single locus in the pig genome to provide a polygene pig genome; (ii) enabling cells containing the polygene pig genome to mature into transgenic pigs. (Item 55) The method according to Item 54, wherein the pig genome is a somatic cell pig genome, the cells are pig zygotes, and the pig zygotes are provided by somatic cell nuclear transfer (SCNT) and transferring the polygene pig genome into the reconstructed SCNT zygote by microinjection. (Item 56) The method according to Item 53, wherein the somatic cell pig genome contains at least one additional genetic modification. (Item 57) The method according to Item 56, wherein the at least one additional genetic modification is selected from the group consisting of gene knockout; gene knock-in; gene replacement; point mutation; deletion, insertion or substitution of a gene, gene fragment or nucleotide; large genomic insertion; or a combination thereof. (Item 58) The method according to Item 55, further comprising introducing at least one additional genetic modification into the polygene pig genome. (Item 59) The method according to Item 56 or 58, wherein the at least one additional genetic modification is selected from the group consisting of gene knockout; gene knock-in; gene replacement; point mutation; deletion, insertion or substitution of a gene, gene fragment or nucleotide; large genomic insertion; or a combination thereof. (Item 60) The method according to item 54, wherein the porcine genome is selected from the group consisting of a germline porcine genome, a zygotic porcine genome, an embryonic porcine genome, or a blastocyst porcine genome. (Item 61) The method according to item 60, wherein the porcine genome comprises at least one further genetic modification. (Item 62) The method according to item 61, wherein the at least one further genetic modification is selected from the group consisting of gene knockout; gene knock-in; gene exchange; point mutation; deletion, insertion or substitution of a gene, gene fragment or nucleotide; large genomic insertion; or a combination thereof. (Item 63) The method according to item 60, further comprising introducing at least one further genetic modification into the polygenic porcine genome. (Item 64) The method according to item 61 or 63, wherein the at least one genetic modification is selected from the group consisting of gene knockout; gene knock-in; gene exchange; point mutation; deletion, insertion or substitution of a gene, gene fragment or nucleotide; large genomic insertion or a combination thereof. (Item 65) The method according to item 54, wherein the integrating comprises a method selected from the group consisting of biological transfection, chemical transfection, physical transfection, virus-mediated transduction or transformation, or a combination thereof. (Item 66) The method according to item 54, wherein the integrating comprises cytoplasmic microinjection and pronuclear microinjection. (Item 67) The method according to item 54, wherein the single locus is a natural locus. (Item 68) The method according to item 54, wherein the single locus is a modified natural locus. (Item 69) The method according to item 68, wherein the modified natural locus comprises a gene editing-mediated insertion or deletion or substitution. (Item 70) The method according to item 68, wherein the modified natural locus contains transgenic DNA. (Item 71) The method according to item 70, wherein the transgenic DNA contains a selectable marker gene. (Item 72) The method according to item 70, wherein the transgenic DNA contains a landing pad. (Item 73) The method according to item 72, wherein the transgenic DNA contains one or more recognition sequences for a polynucleotide modifying enzyme. (Item 74) The method according to item 73, wherein the polynucleotide modifying enzyme is selected from the group consisting of engineered endonucleases, site-specific recombinases, integrases, or combinations thereof. (Item 75) The method according to item 74, wherein the engineered endonuclease is selected from the group consisting of zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats / Cas9 nuclease. (Item 76) The method according to item 75, wherein the site-specific recombinase is selected from the group consisting of lambda integrase, Cre recombinase, FLP recombinase, gamma-delta resolvase, Tn3 resolvase, ΦC31 integrase, Bxb1-integrase, R4 integrase, or combinations thereof. (Item 77) The method according to item 54, wherein the single locus is a natural GGTA1 locus. (Item 78) The method according to item 54, wherein the single locus is a modified GGTA1 locus. (Item 79) The method according to item 78, wherein the modified GGTA1 locus is a transgenic GGTA1 locus. (Item 80) The method according to item 79, wherein the transgenic GGTA1 locus contains a selectable marker gene. (Item 81) The method according to item 79, wherein the transgenic GGTA1 locus comprises an engineered landing pad. (Item 82) The method according to item 54, wherein the single locus is a natural locus selected from the group consisting of CMAH, β4GalNT2, AAVS1 locus and Rosa26. (Item 83) The method according to item 54, wherein the single locus is a modified locus selected from the group consisting of CMAH, β4GalNT2, AAVS1 locus and Rosa26. (Item 84) The method according to item 56 or 61, wherein the single locus is not the GGTA1 locus and the further genetic modification comprises knocking out the alpha1,3-galactosyltransferase gene. (Item 85) The method according to item 56, 58, 61 or 63, wherein the at least one further genetic modification comprises the integration and expression of CD46. (Item 86) The method according to item 56, 58, 61 or 63, wherein the at least one further genetic modification comprises the integration and expression of human HLA-E. (Item 87) The method according to item 56, 58, 61 or 63, wherein the at least one further genetic modification comprises the modification of the porcine vWF locus to reduce or eliminate spontaneous aggregation of human platelets. (Item 88) The method according to item 56, 58, 61 or 63, wherein the at least one further genetic modification comprises the integration and expression of chimeric porcine-human vWF. (Item 89) The method according to item 56, 58, 61 or 63, wherein the at least one further genetic modification comprises targeted inactivation of the porcine vWF gene and insertion and expression of a fragment of the human vWF gene. (Item 90) The method according to item 56, 58, 61 or 63, wherein the at least one further genetic modification comprises a modification of porcine vWF produced by nucleotide substitution within the native vWF gene or by knockout of the native vWF gene and replacement with a full human vWF gene. (Item 91) The method according to item 56, 58, 61 or 63, wherein the at least one genetic modification comprises knockout of a gene selected from the group consisting of the β4GalNT2 gene, the CMAH gene, the β4GalNT2 gene and the GGTA1 gene. (Item 92) A transgenic animal or production herd produced by the method according to item 54 or 61. (Item 93) The method according to item 54 or 60, further comprising mating the transgenic pig with a second transgenic pig, wherein the second transgenic pig comprises at least one genetic modification. (Item 94) The method according to item 93, wherein the at least one genetic modification comprises integration and expression of at least one transgene. (Item 95) The method according to item 94, wherein the at least one transgene is selected from the group consisting of anticoagulation factors, complement inhibitors, immunomodulatory factors, cytoprotective transgenes and combinations thereof. (Item 96) The method according to item 93, wherein the at least one genetic modification comprises knockout of at least one porcine gene. (Item 97) The method according to item 93, wherein the at least one genetic modification comprises integration and expression of chimeric porcine-human vWF. (Item 98) The method according to item 94, wherein the at least one further genetic modification comprises modification of the porcine vWF locus to reduce or eliminate spontaneous aggregation of human platelets. (Item 99) The method according to item 98, wherein the modified porcine vWF is produced by nucleotide substitution within the native vWF gene or by knockout of the native vWF gene and replacement with the complete human vWF gene. (Item 100) A transgenic animal or production group produced by the method according to item 94. (Item 101) A method for treating a subject in need thereof, the method comprising transplanting into the subject at least one organ, organ fragment, tissue or cell derived from the transgenic pig according to items 1 to 53. (Item 102) The method according to item 101, wherein the at least one organ is selected from the group consisting of the lung, heart, kidney, liver, pancreas or combinations thereof. (Item 103) The method according to item 102, wherein the at least one organ is the lung. (Item 104) The method according to item 101, wherein the at least one organ fragment is selected from lung fragments, heart fragments, kidney fragments, pancreas fragments or combinations thereof. (Item 105) The method according to item 101, wherein the subject has a progressive lung disease and a lung or lung fragment is transplanted. (Item 106) The method according to item 105, wherein the progressive lung disease is associated with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPD), cystic fibrosis (CF), alpha1-antitrypsin disease or primary pulmonary hypertension. (Item 107) The method according to item 101, further comprising administering to the subject one or more therapeutic agents. (Item 108) The method according to item 107, wherein the therapeutic agent is selected from antirejection agents, anti-inflammatory agents, immunosuppressive agents, immunomodulatory agents, antibacterial agents and antiviral agents, and combinations thereof. (Item 109) A transgenic pig containing a genetic modification at the porcine vWF locus and lacking the expression of alpha 1,3 galactosyltransferase. (Item 110) The transgenic pig according to item 109, further comprising at least one additional genetic modification. (Item 111) The transgenic pig according to item 110, wherein the at least one additional genetic modification is selected from the group consisting of gene knockout; gene knock-in; gene replacement; point mutation; deletion, insertion or substitution of a gene, gene fragment or nucleotide; large genomic insertion and combinations thereof. (Item 112) The transgenic pig according to item 110, wherein the at least one additional genetic modification comprises the integration and expression of at least four transgenes.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0085] The present invention is directed to transgenic animals that are particularly useful as donors of organs, organ fragments, tissues or cells for xenotransplantation. In particular, the present invention is directed to transgenic ungulates, more particularly transgenic pig animals (pigs), that are useful as donors of organs, organ fragments, tissues or cells for xenotransplantation. The present invention also extends to organs, organ fragments, tissues or cells derived from such donor animals, methods of producing such donor animals, and the use of organs, organ fragments, tissues or cells derived from such animals in the treatment of diseases and disorders.

[0086] Conveniently, the donor animals provide organs, organ fragments, tissues and cells that are functionally superior for transplantation (tx) compared to organs, organ fragments, tissues and cells known in the art. Without wishing to be bound by any particular theory, the organs, organ fragments, tissues and cells of the present invention are believed to have improved survival and / or functionality for the notable reduction of consumptive coagulopathy (also known as disseminated intravascular coagulation (DIC)) and thrombotic microangiopathy currently observed after mismatched xenotransplantation.

[0087] The organ or organ fragment may be any suitable organ, such as, for example, the lung, heart, liver or pancreas. The tissue may be any suitable tissue, such as, for example, epithelial or connective tissue. The cell may be any suitable cell. The cell may be any suitable cell, such as, for example, islet cells.

[0088] In an exemplary embodiment, the present invention provides transgenic animals (e.g., ungulates, porcine animals) that are particularly useful as a source of organs (i.e., lungs), organ fragments, tissues or cells for lung xenotransplantation, organs (i.e., lungs), organ fragments, tissues and cells derived therefrom, and methods of producing the transgenic animals, and methods of using the organs, tissues and cells derived therefrom for lung xenotransplantation.

[0089] Conveniently, organs, organ fragments, tissues or cells derived from transgenic animals result in low levels of one or more of the following, or none at all, after xenotransplantation: hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR / DXR) and / or acute cellular xenograft rejection (ACXR).

[0090] In one embodiment, organs, organ fragments, tissues or cells derived from transgenic animals result in low levels of HAR and AHXR, or none at all, after xenotransplantation. In another embodiment, organs, organ fragments, tissues or cells derived from transgenic animals result in low levels of HAR, AHXR and ACXR, or none at all, after xenotransplantation.

[0091] In an exemplary embodiment, the transgenic animal lacks any expression of functional alpha1,3-galactosyltransferase (alphaGal) (as a result of genetic modification or otherwise), and incorporates at least some additional genetic modifications (e.g., gene knockout, gene knock-in, gene replacement, point mutation, deletion, insertion or substitution (i.e., of a gene, gene fragment or nucleotide), large genomic insertion or combinations thereof), and is a swine animal. The genetic modification may be mediated by any suitable technique, including, for example, homologous recombination or gene editing methods.

[0092] In an exemplary embodiment, the transgenic animal lacks any expression of functional alpha1,3-galactosyltransferase (alphaGal) (as a result of genetic modification or otherwise), and incorporates and expresses at least four transgenes under the control of at least two promoters at a single locus, and is a porcine animal. In certain embodiments, one promoter controls the expression of one transgene, e.g., the expression of each of the at least four transgenes is controlled by a single (dedicated) promoter. In alternative embodiments, one promoter controls the expression of more than one transgene, e.g., one promoter controls the expression of two transgenes. Conveniently, during mating, four or more transgenes are incorporated together, co-expressed, and segregate together. The single locus may be different. In certain embodiments, the single locus is a natural or modified natural locus. The modified natural locus can be modified by any suitable technique including, but not limited to, CRISP-induced insertion or deletion (indel), introduction of a selectable marker gene (e.g., neo), or introduction of a large genomic insertion fragment (e.g., landing pad) for the purpose of facilitating the incorporation of one or more transgenes. In certain embodiments, the single locus is a natural or modified GGTA1 locus. The GGTA1 locus is inactivated by the incorporation and expression of at least four transgenes, e.g., by application of homologous recombination, gene editing, or recombinase technology. The single locus may be, for example, AAVS1, ROSA26, CMAH, or β4GalNT2. Optionally, the transgenic animal can have one or more additional genetic modifications and / or the expression of one or more additional porcine genes can be modified by mechanisms other than genetic modification.

[0093] In an exemplary embodiment, the transgenic animal lacks any expression of functional alpha1,3-galactosyltransferase (alphaGal) (as a result of genetic modification or otherwise), and incorporates and expresses at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 transgenes or more at a single locus, and is a pig animal. In certain embodiments, the expression of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 transgenes or more is controlled by at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 promoters or more. In certain embodiments, the promoter is specific to the transgene, i.e., one promoter controls the expression of one transgene, but in alternative embodiments, one promoter controls the expression of more than one transgene, for example, one promoter controls the expression of two transgenes. Conveniently, during mating, two or more additional transgenes are incorporated together, co-expressed, and segregate together. The single locus may be different. In certain embodiments, the single locus is a natural or modified natural locus. The modified natural locus can be modified by any suitable technique including, but not limited to, CRISP-induced insertion or deletion (indel), introduction of a selectable marker gene (e.g., neo), or introduction of a large genomic insertion fragment (e.g., landing pad) for the purpose of facilitating the integration of one or more transgenes. In certain embodiments, the single locus is a natural or modified GGTA1 locus. The GGTA1 locus is inactivated by the integration and expression of at least 4 transgenes, for example, by the application of homologous recombination, gene editing, or recombinase technology. The single locus may be, for example, AAVS1, ROSA26, CMAH, or β4GalNT2.Optionally, the donor animal can have further genetic modifications and / or the expression of one or more additional porcine genes can be modified by mechanisms other than genetic modification.

[0094] In an exemplary embodiment, the transgenic animal lacks any expression of functional alpha1,3-galactosyltransferase (alphaGal) (as a result of genetic modification or otherwise), incorporates and expresses at least four transgenes at a single locus (i.e., locus 1), incorporates and expresses one or more additional transgenes at a second single locus (i.e., locus 2), and is a pig animal. In certain embodiments, one promoter controls the expression of one transgene, and for example, the expression of each of at least four transgenes at locus 1 or locus 2 is controlled by a single (dedicated) promoter. In alternative embodiments, one promoter controls the expression of more than one transgene, and for example, one promoter controls the expression of two transgenes at locus 1. Specific loci may vary. In certain embodiments, the first single locus is GGTA1 and the second single locus is, for example, CMAH, B4GalNT2, or vWF. In certain embodiments, at least four transgenes are incorporated and expressed at each single locus, i.e., locus 1 and locus 2, to produce an animal having eight or more transgenes expressed at two different independent loci. In certain embodiments, the single locus is a natural or modified natural locus. The modified natural locus can be modified by any suitable technique including, but not limited to, CRISP-induced insertion or deletion (indel) to facilitate the incorporation of one or more transgenes, introduction of a selectable marker gene (e.g., neo), or introduction of a large genomic insertion fragment (e.g., landing pad). Optionally, the donor animal can have further genetic modifications and / or the expression of one or more additional pig genes can be modified by mechanisms other than genetic modification. Conveniently, during mating, two or more additional transgenes are incorporated together, co-expressed, and segregate together.

[0095] At least two promoters may be different. The promoter may be exogenous or natural. In an exemplary embodiment, the promoter is constitutive or regulatable (e.g., tissue-specific, inducible). In one embodiment, both promoters may be constitutively or ubiquitously expressed in the donor animal (e.g., from a CAG or similar promoter). In another embodiment having two promoters, one promoter will allow for tissue-specific expression of the transgene (e.g., endothelial-specific expression), while the second promoter will allow for constitutive or ubiquitous expression of one or more transgenes (at the same integration site) (e.g., from a CAG or similar promoter).

[0096] In certain embodiments, additional genetic modifications (i.e., separate from the integration and expression of the multiple transgenes described above) can result in the inactivation of specific porcine genes, including but not limited to the porcine von Willebrand factor (vWF) gene, or the replacement of part or all of the porcine vWF gene with an equivalent counterpart from the human vWF gene. Other genes that can be inactivated in relation to additional genetic modifications include, for example, CMP-NeuAc hydroxylase (CMAH), isogloboside 3 synthase, β4Gal, NT2 Forssman synthase, or combinations thereof. In certain embodiments, the single locus for transgene integration is not GGTA1, and the additional genetic modification includes the inactivation of GGTA1.

[0097] In certain embodiments, the additional genetic modification is, for example, a gene editing-induced deletion / insertion or gene replacement (INDEL).

[0098] In certain embodiments, additional genetic modifications (i.e., separate from the integration and expression of the multiple transgenes described above) can result in the integration and expression of one or more transgenes at a second locus.

[0099] In one embodiment, the invention is a porcine animal that lacks any expression of functional alpha 1,3 galactosyltransferase (alphaGal) (as a result of genetic modification or otherwise), and further comprises inactivation of the porcine von Willebrand factor (vWF) gene, or partial or complete replacement of the porcine vWF gene with an equivalent counterpart from the human vWF gene. Optionally, the porcine animal comprises one or more additional genetic modifications. In certain embodiments, the animal can be mated with a second animal having one or more genetic modifications.

[0100] The invention also extends to methods of making and using such transgenic animals (or organs, tissues or cells derived therefrom).

[0101] In an exemplary embodiment, the invention is a method of producing a transgenic pig that expresses at least four transgenic genes but lacks expression of alpha 1,3 galactosyltransferase, the method comprising: (i) integrating at least four transgenes under the control of at least two promoters at a single locus in the porcine genome to provide a polygenic porcine genome; and (ii) enabling cells comprising the polygenic porcine genome to mature into a transgenic pig.

[0102] In certain embodiments, the porcine genome is a somatic porcine genome and the cells are porcine zygotes.

[0103] In certain embodiments, the porcine genome is selected from the group consisting of a germline porcine genome, a zygotic porcine genome, an embryonic porcine genome or a blastocyst porcine genome.

[0104] In an exemplary embodiment, integrating comprises a method selected from the group consisting of biological transfection, chemical transfection, physical transfection, viral-mediated transduction or transformation, or combinations thereof.

[0105] In certain embodiments, incorporation includes cytoplasmic microinjection and pronuclear microinjection.

[0106] In exemplary embodiments, the method includes the use of dicistronic or polycistronic vectors that have functional and / or production advantages that allow for the co-incorporation and co-expression of transgenes, including multicistronic vectors that utilize 2A technology. In preferred embodiments, each dicistron in a polycistronic vector containing at least four transgenes is under the control of its own promoter, and one or both of the promoters may result in constitutive expression of two or more genes, and the second promoter may result in tissue-specific expression of two or more genes. These vectors are utilized in combination with gene editing tools including editing nucleases and / or site-specific integrases.

[0107] The invention also extends to methods of treating a subject in need thereof with one or more organs, organ fragments, tissues or cells derived from the transgenic animals of the invention. In exemplary embodiments, the organ is a liver, lung, heart, pancreas or other solid organ. Examples of tissues contemplated by the invention include, without limitation, epithelial and connective tissues.

[0108] Grafts comprising more than one organ or organ fragment are also contemplated by the invention. For example, a graft comprising a lung (or a fragment of a lung) and a heart (or a fragment thereof) is contemplated by the invention.

[0109] Definitions As used herein, the term "adverse event" refers to any undesirable or unintended sign (including abnormal test findings), symptom or disease that is temporarily associated with the use of a medical product (e.g., a xenograft), whether or not it is considered to be related to the medical product.

[0110] As used herein, the term "animal" refers to a mammal. In a specific embodiment, the animal is at least 6 months old. In certain embodiments, the animal is past the weaning age. In certain embodiments, the animal survives until it reaches breeding age. The animals of the present invention are "genetically modified" or "transgenic", which means that in at least one cell of the animal and generally to mediate a genotypic or phenotypic effect on at least one germ cell of the animal, they have a transgene or other foreign DNA that is added or integrated, or a modified endogenous gene that includes targeting, recombination, disruption, deletion, destruction, exchange, suppression, enhancement or otherwise alteration. In some embodiments, the animal can have a transgene incorporated into one allele of its genome (heterozygous transgenic). In other embodiments, the animal can have a transgene on both alleles (homozygous transgenic).

[0111] As used herein, the term "mating" or "mated" or derivatives thereof refers to any means of reproduction, including both natural and artificial means.

[0112] As used herein, the terms "mating colony" or "production colony" refer to a group of transgenic animals generated by the methods of the present invention. In some embodiments, the genetic modifications can be identified in animals that are then mated together to form a group of animals having a desired set of genetic modifications (or a single genetic modification). See WO2012 / 112586; PCT / US2012 / 025097. These progeny can be further mated to produce different or the same set of genetic modifications (or a single genetic modification) in their progeny. This mating cycle for animals having the desired genetic modification(s) can be continued as desired. In this context, a "colony" can include multiple generations of animals produced over a long period of time having the same or different genetic modification(s). A "colony" can also refer to a single generation of animals having the same or different genetic modification(s).

[0113] As used herein, the terms "CRISPR" or "clustered regularly interspaced short palindromic repeats" or "SPIDR" or "spacer interspersed direct repeats" refer to a family of DNA loci that are typically specific to certain bacterial species. CRISPR loci contain different classes of short, interspersed repeats (SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433

[1987] ; and Nakata et al., J. Bacteriol., 171:3553-3556

[1989] ) and related genes. CRISPR / Cas molecules are components of a prokaryotic adaptive immune system that is functionally similar to eukaryotic RNA interference, using RNA base pairing to direct DNA or RNA cleavage. Inducing DNA DSBs requires two components: the Cas9 protein, which functions as an endonuclease, and the CRISPR RNA (crRNA) and tracer RNA (tracrRNA) sequences that help direct the Cas9 / RNA complex to the target DNA sequence (Makarova et al., Nat Rev Microbiol, 9(6) : pp. 467-477, 2011). Modifying a single targeting RNA may be sufficient to change the nucleotide target of the Cas protein. In some cases, the crRNA and tracrRNA can be engineered as a single cr / tracrRNA hybrid that leads to Cas9 cleavage activity (Jinek et al., Science, 337(6096):816-821, 2012). As described elsewhere, the CRISPR / Cas system can be used in bacteria, yeast, humans, and zebrafish (e.g., Jiang et al., Nat Biotechnol, 31(3):233-239, 2013; Dicarlo et al., Nucleic Acids Res, doi:10.1093 / nar / gkt135, 2013; Cong et al., Science, 339(6121):819-823, 20 13; Mali et al., Science, 339(6121):823-826, 2013; Cho et al., Nat Biotechnol, 31(3):230-232, 2013; and Hwang et al., Nat Biotechnol, 31(3):227-229, 2013).

[0114] As used herein, the term "clinically appropriate immunosuppressive regimen" refers to a clinically acceptable regimen of immunosuppressive agents provided to a patient following transplantation of an organ, tissue, or cell of a genetically modified pig disclosed herein. Determining clinical appropriateness generally requires a determination by the FDA of a balance of acceptable risks to potential benefits such that human safety is maintained while the efficacy of the drug or treatment is maintained.

[0115] As used herein, the term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes production of the gene product in a cell under most or all physiological conditions of the cell.

[0116] As used herein, the term "donor" includes any non-human animal that can serve as a source of donor organs, tissues, or cells for xenotransplantation. The donor may be at any stage of development, including but not limited to fetus, neonate, juvenile, and adult.

[0117] As used herein, the term "endogenous" when used in reference to a nucleic acid sequence and an animal refers to any nucleic acid sequence that is naturally present in the genome of that animal. Endogenous nucleic acid sequences can include one or more gene sequences, intergenic sequences, portions of gene sequences or intergenic sequences, or combinations thereof.

[0118] As used herein, terms such as "endothelium-specific", "specific transgene expression in endothelial tissue", "specifically expressing at least one transgene in endothelial tissue", and the like are understood to refer to a transgene under the control of an endothelium-specific regulatory element that enables limited expression of the transgene in endothelial tissue and / or cells. The function and expression of the transgene are limited to endothelial tissue and / or cells.

[0119] As used herein, the term "endothelium" is an epithelium of mesodermal origin composed of a single layer of thin, flat cells that line the inner side of internal body cavities. For example, the serous cavities or the interior of the heart contain an endothelial cell lining, and "vascular endothelium" is the endothelium that lines the inside of blood vessels.

[0120] As used herein, terms such as "endothelium-specific regulatory element" refer to a promoter, enhancer, or combination thereof, where the promoter, enhancer, or combination thereof drives limited expression of a transgene in endothelial tissue and / or cells. The regulatory element provides for the function and expression of a transgene that is limited to endothelial tissue and / or cells.

[0121] As used herein, the term "enhancer" refers to an element in a nucleic acid construct that is intended to promote, in a tissue-specific manner, an increase in the expression of a transgene. An enhancer is an external element that significantly alters the efficiency of gene transcription (Molecular Biology of the Gene, 4th Edition, pages 708 - 710, Benjamin Cummings Publishing Company, Menlo Park, CA (C) 1987). In certain embodiments, an animal expresses a transgene under the control of a promoter in combination with an enhancer element. In some embodiments, the promoter is used in combination with an enhancer element that is a non-coding or intron region of DNA that is substantially associated with or co-localized with the promoter.

[0122] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts), and / or the subsequent process by which the transcribed mRNA is translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as a "gene product". When the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in eukaryotic cells.

[0123] As used herein, the term "gene" is used broadly to refer to any segment of DNA associated with a biological function. Thus, a gene includes coding sequences and / or regulatory sequences required for their expression. A gene can also include non-expressed DNA segments that form recognition sequences for other proteins, for example. A gene can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and can include sequences designed to have desired parameters.

[0124] As used herein, the term "gene editing" refers to a type of genetic manipulation that uses gene editing tools to insert, exchange, or remove DNA from the genome. Examples of gene editing tools include, without limitation, zinc finger nucleases, TALENs, and CRISPRs.

[0125] As used herein, the term "gene editing agent" or similar terms refer to the modification of genes (e.g., deletions, substitutions, rearrangements) that involve the use of gene editing / gene editing tools.

[0126] As used herein, the term "gene knockout" refers to a genetic modification resulting from disruption of the genetic information encoded at a chromosomal locus.

[0127] As used herein, the term "gene knock-in" is a genetic modification resulting from the exchange of the genetic information encoded at a chromosomal locus with a different DNA sequence.

[0128] The term "genetic modification," as used herein, refers to one or more alterations of nucleic acids, e.g., nucleic acids in the genome of an organism. For example, genetic modification can refer to changes in genes, additions (e.g., gene knock-ins), and / or deletions (e.g., gene knockouts).

[0129] As used herein, the term "high" with respect to the level of expression refers to an expression level that is considered sufficient to provide a phenotype (detectable expression or therapeutic benefit). Generally, a "high" expression level is sufficient to be able to reduce graft rejection, including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), T cell-mediated cellular rejection, and immediate blood-mediated inflammatory response (IBMIR).

[0130] As used herein, the terms "homology-directed recombination" or "homology-directed repair" or "HDR" are used to refer to homologous recombination events initiated by the presence of a double-strand break (DSB) in DNA (Liang et al. 1998); the specificity of HDR can be controlled when combined with any genome editing technology known to generate highly efficient targeted double-strand breaks and to enable precise editing of the genome of the targeted cells; for example, the CRISPR / Cas9 system (Findlay et al., 20 14; Mali et al., February 2014; and Ran et al., 2013).

[0131] As used herein, the term "enhanced homology-directed insertion or knock-in" refers to homology-directed recombination combined with any genome editing technology known to generate highly efficient targeted double-strand breaks and to enable precise editing of the genome of the targeted cells; for example, as described for the insertion of a DNA construct, more specifically a large DNA fragment or construct flanked by homology arms or segments of DNA homologous to the double-strand break, using the CRISPR / Cas9 system. (Mali et al., February 2013).

[0132] As used herein, the term "humanized" refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) includes portions that correspond substantially or identically to the structure of a particular gene or protein found in a non-human animal, and differs from that found in the relevant particular non-human gene or protein, and instead includes portions that more closely correspond to the equivalent structure found in the corresponding human gene or protein. In some embodiments, a "humanized" gene encodes a polypeptide having an amino acid sequence such as that of a substantially human polypeptide (e.g., a human protein or a portion thereof, e.g., a characteristic portion thereof). The term "hyperacute rejection" refers to the rejection of a transplanted material or tissue that occurs or begins within the first 24 hours after transplantation.

[0133] As used herein, the terms "implant", "transplant", or "graft" are understood to refer to the act of inserting a tissue or organ into a subject under conditions that allow for angiogenesis in the tissue or organ; and also refer to the tissue or organ so inserted (i.e., "implanted", "transplanted", or "grafted"). Conditions that favor angiogenesis of a graft in a mammal include a localized tissue bed having an extensive blood supply network at the site of implantation.

[0134] As used herein, the term "immunomodulatory factor" refers to a transgene having the ability to modulate an immune response. In an exemplary embodiment, the immunomodulatory factor according to the present invention may be a complement inhibitory factor or an immunosuppressive factor. In a specific embodiment, the immunomodulatory factor is a complement inhibitory factor. The complement inhibitory factor may be CD46 (or MCP), CD55, CD59, and / or CRI. In a specific embodiment, at least two complement inhibitory factors can be expressed. In one embodiment, the complement inhibitory factors may be CD55 and CD59. In another embodiment, the immunomodulatory factor may be a class II transactivator or a mutant thereof. In certain embodiments, the immunomodulatory factor may be a class II transactivator dominant negative mutant (CIITA-DN). In another specific embodiment, the immunomodulatory factor is an immunosuppressive factor. The immunosuppressive factor may be CTLA4-Ig. Other immunomodulatory factors include, but are not limited to, CIITA-DN, PDL I, PDL2, or tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL, CD95L), CD47 known as integrin-associated protein (CD47), HLA-E, HLA-DP, HLA-DQ, and / or HLA-DR, and can be selected from the group consisting of these.

[0135] As used herein, an "inducible" promoter is a promoter that is under environmental or developmental regulation.

[0136] As used herein, the term "landing pad" or "engineering operation landing pad" refers to a nucleotide sequence containing at least one recognition sequence that is selectively bound or modified by a specific polynucleotide-modifying enzyme such as a site-specific recombinase and / or a targeting endonuclease. Generally, the recognition sequence(s) in the landing pad sequence do(es) not exist endogenously in the genome of the cell to be modified. The targeting integration rate can be improved by selecting a recognition sequence for a highly efficient polynucleotide-modifying enzyme that does not exist endogenously in the genome of the targeted cell. The selection of a recognition sequence that does not exist endogenously also reduces potential off-target integration. In other embodiments, the use of a recognition sequence specific to the cell to be modified may be desirable. For example, when multiple recognition sequences are used in the landing pad sequence, one or more may be exogenous and one or more may be specific. Multiple recognition sequences may be present in a single landing pad, allowing sequential targeting of the landing pad by two or more polynucleotide-modifying enzymes so that two or more unique sequences can be inserted. Alternatively, the presence of multiple recognition sequences in the landing pad allows insertion of multiple copies of the same sequence into the landing pad. The landing pad can comprise at least one recognition sequence. For example, an exogenous nucleic acid can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, or more recognition sequences. In embodiments comprising more than one recognition sequence, the recognition sequences may be specific to each other (i.e., recognized by different polynucleotide-modifying enzymes), the same repeat sequence, or a combination of repeat and specific sequences. Optionally, the landing pad can comprise one or more sequences encoding a selectable marker such as an antibiotic resistance gene, a metabolic selection marker, or a fluorescent protein.Other sequences, such as transcriptional regulatory and control elements (i.e., promoters, partial promoters, promoter traps, start codons, enhancers, introns, insulators and other expression elements), may be present.

[0137] As used herein, the term "large targeting vector" or "LTVEC" includes large targeting vectors for eukaryotic cells, derived from fragments of cloned genomic DNA that are larger than those commonly used by other approaches aimed at performing homologous gene targeting in eukaryotic cells. Examples of LTVECs include, but are not limited to, bacterial artificial chromosomes (BACs), human artificial chromosomes (HACs) and yeast artificial chromosomes (YACs).

[0138] As used herein, the term "genomic locus" or "locus" (plural loci) is the specific position of a gene or DNA sequence on a chromosome and can include both intron and exon sequences of a particular gene. A "gene" refers to a stretch of DNA or RNA that encodes a polypeptide or has a functional role to play in an organism and is thus the molecular unit of heredity in a living organism. In the present invention, a gene can be considered to include regions that regulate the production of a gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcribed sequences. Thus, a gene includes, but is not necessarily limited to, introns, exons, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, 5' or 3' regulatory sequences, origins of replication, matrix attachment sites and locus control regions.

[0139] As used herein, the term "lung transplantation" refers to a surgical procedure in which a patient's diseased lungs are partially or completely replaced with lungs from a donor. Lung transplantation may be "single," where only one of the two lungs in the recipient is removed and replaced with a single lung from the donor, or "bilateral," which involves removing both lungs, one on each side, and replacing them with both lungs from the donor. In certain embodiments, the lungs are transplanted together with the heart.

[0140] As used herein, the term "lung preservation" refers to the process of maintaining and protecting donor lungs from the time of lung procurement until transplantation in the recipient.

[0141] As used herein, the phrase "loss of graft function" refers to any physiological disruption or dysfunction of the normal processes exhibited by an organ or tissue in a donor animal, as used herein.

[0142] As used herein, the term "mammal" refers to any non-human mammal, including but not limited to pigs, sheep, goats, cows (Bos genus), deer, rabbits, horses, monkeys, dogs, cats, rats, and mice. In certain embodiments, the animal is a pig animal of at least 300 pounds. In a specific embodiment, the mammal is a female pig that has given birth at least once. In certain embodiments, the mammal is a non-human primate, such as a monkey or baboon.

[0143] As used herein, a "marker" or "selectable marker" is a selectable marker that enables the isolation of rare transfected cells that express the marker from the majority of treated cells in a population. Such marker genes include, but are not limited to, neomycin phosphotransferase and hygromycin B phosphotransferase, or fluorescent proteins, such as GFP.

[0144] As used herein, the terms "nucleotide", "polynucleotide", "nucleotide sequence", "nucleic acid", and "oligonucleotide" are used interchangeably. They refer to polymers of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: the coding or non-coding regions of genes or gene fragments, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The term also encompasses nucleic acid-like structures having a synthetic backbone, for example, Eckstein, 1991; Baserga et al., 1992; Milligan, 1993 ; see WO97 / 03211; WO96 / 39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide can include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Where present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Non-nucleotide constituents can be interspersed within the nucleotide sequence. A polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling constituent.

[0145] As used herein, the phrase "operatively linked" includes the relationship of operatively linked components that function in their intended manner. In one example, a nucleic acid sequence encoding a protein can be operatively linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.) so as to maintain appropriate transcriptional regulation.

[0146] As used herein, the term "organ" refers to a collection of tissues that are joined into a structural unit to perform a common function. The organ may be a solid organ. A solid organ is an internal organ that has a firm tissue consistency and is neither hollow (such as the organs of the gastrointestinal tract) nor liquid (such as blood). Examples of solid organs include the heart, kidney, liver, lung, pancreas, spleen, and adrenal gland.

[0147] As used herein, the term "primate" refers to various mammals of the order Primates, including apes, including fox monkeys, lorises, tarsiers, New World monkeys, Old World monkeys, and humans, and is characterized by nails on the hands and feet, a short nose, and a large brain. In certain embodiments, the primate is a non-human primate. In other embodiments, the primate is a human.

[0148] As used herein, the term "promoter" generally refers to a region of DNA upstream (5') of a coding region, which at least partially controls the initiation and level of transcription. References to "promoter" herein should be taken in the broadest context and include not only the transcriptional regulatory sequences of classical genomic genes, including TATA box or non-TATA box promoters, but also additional regulatory elements (i.e., activation sequences, enhancers and silencers) that alter gene expression in response to developmental and / or environmental stimuli, or in a tissue-specific or cell-type specific manner. A promoter is usually, but not necessarily, located upstream or 5' of the structural gene whose expression it regulates. Further, regulatory elements including a promoter are usually located within 2 kb of the transcriptional start site of a gene, although they may be located several kb away. A promoter can contain additional specific regulatory elements located further away from the start site in order to further enhance expression in a cell and / or to alter the timing or inducibility of expression of the structural gene to which it is operably linked.

[0149] As used herein, the terms "swine", "swine animal", "pig" and "hog" are generic terms that refer to animals of the same species regardless of sex, size or breed.

[0150] As used herein, the term "recognition site" or "recognition sequence" refers to a specific DNA sequence that is recognized by a nuclease or other enzyme, binds to the DNA backbone, and leads to site-specific cleavage.

[0151] As used herein, the term "recombination site" refers to a nucleotide sequence that is recognized by a site-specific recombinase and can serve as a substrate for a recombination event.

[0152] As used herein, the terms "regulatory element" and "expression control element" are used interchangeably and refer to nucleic acid molecules that can affect the transcription and / or translation of a coding sequence operably linked in a particular context. These terms are used in a broad sense and include all elements that promote or regulate transcription, including core elements, upstream elements, enhancers and response elements required for the basic interactions of promoters, RNA polymerases and transcription factors. (See, e.g., Lewin, "Genes V" (Oxford University Press, Oxford), pp. 847-873). Exemplary regulatory elements in prokaryotes include promoters, operator sequences and ribosome binding sites. Regulatory elements used in eukaryotic cells may include, without limitation, promoters, enhancers, splicing signals and polyadenylation signals.

[0153] As used herein, the term "regulatable promoter" refers to a promoter that can be used to regulate whether a peptide is expressed in an animal, tissue or organ. A regulatable promoter can be tissue-specific, capable of being expressed in only a particular tissue, or temporarily regulatable (switched on at a particular time (operating according to the stage of development)), or inducible, such that it is simply switched on or off (expressed or not expressed) when controlled by an inducible element. (It may be an inducible promoter, such as an immune-inducible promoter and a cytokine response promoter, and may be induced, for example, by interferon gamma, TNF-alpha, IL-1, IL-6 or TGF-beta). For example, expression can be blocked while an organ or tissue is part of a pig, but induced if the pig is transplanted into a human for some time to overcome the cellular immune response. Furthermore, the expression level can be controlled by a regulatable promoter system to ensure that immunosuppression of the recipient's immune system does not occur.

[0154] As used herein, the terms "regulatory sequence", "regulatory element" and "control element" are interchangeable and refer to polynucleotide sequences that are upstream (5' non-coding sequence), within or downstream (3' untranslated sequence) of the polynucleotide target to be expressed. Regulatory sequences affect, for example, the timing of transcription, the amount or level of transcription, RNA processing or stability and / or the translation of the associated structural nucleotide sequences. Regulatory sequences can include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translation initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites and the like.

[0155] As used herein, the term "safe harbor" locus refers to a site in the genome into which transgenic DNA (e.g., a construct) can be added without harm and which can produce a consistent level of expression. In certain embodiments, the invention includes the integration and expression of transgenic DNA containing a transgene in a safe harbor locus.

[0156] As used herein, the term "site-specific recombinase" refers to a group of enzymes that can promote recombination between "recombination sites" where two recombination sites are physically separated within a single nucleic acid molecule or on separate nucleic acid molecules. Examples of "site-specific recombinases" include, but are not limited to, phiC31, att, Bxb1, R4 (integrase) and / or Cre, Flp and Dre recombinases.

[0157] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc. (e.g., those that will become recipients of a particular treatment (e.g., transplantation of a graft) or those that are donors of a graft). Unless otherwise specified herein (e.g., if the subject is a graft donor), the terms "subject" and "patient" are used interchangeably with respect to human subjects.

[0158] As used herein, the term "targeting vector" refers to a recombinant DNA construct that generally contains DNA arms that are homologous to genomic DNA adjacent to critical elements of a target gene or target sequence. When introduced into a cell, the targeting vector is incorporated into the cell genome through homologous recombination. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes production of the gene product in a cell only when the cell is substantially of the tissue type corresponding to the promoter.

[0159] As used herein, the term "tissue" refers to the cellular tissue level intermediate between cells and a complete organ. A tissue is an ensemble of similar cells from the same origin that perform a particular function together. Then, organs are formed by bringing together multiple tissues by function. Examples of tissues contemplated by the present invention include, but are not limited to, connective tissue, muscle tissue, nerve tissue, epithelial tissue, and petrified tissue. Blood, bone, tendon, ligament, fat, and areolar tissue are examples of connective tissue, which can also be classified as fibrous connective tissue, skeletal connective tissue, and fluid connective tissue. Muscle tissue is separated into three different categories: visceral or smooth muscle, found in the inner lining of organs; skeletal muscle, generally attached to bone and causing movement of the whole body; and cardiac muscle, found in the heart and contracting to pump blood throughout the organism. Cells that make up the central nervous system and the peripheral nervous system are classified as nervous (or nerve) tissue. In the central nervous system, nerve tissue forms the brain and spinal cord. In the peripheral nervous system, nerve tissue forms the cranial nerves and spinal nerves, which include motor neurons.

[0160] The term transcription activator-like effector nuclease or "TALEN", as used herein, refers to an artificial restriction enzyme generated by fusing a TAL effector DNA binding domain to a DNA cleavage domain. These reagents enable efficient, programmable, and specific DNA cleavage and are powerful tools for in situ genome editing. Transcription activator-like effectors (TALEs) can be readily engineered to bind to almost any DNA sequence. The term TALEN, as used herein, is broad and includes monomeric TALENs that can cleave double-stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together can be referred to as left TALEN and right TALEN, which refers to the left and right images of DNA. See U.S. Patent Serial Number 12 / 965,590; U.S. Patent Serial Number 13 / 426,991 (U.S. Patent No. 8,450,471); U.S. Patent Serial Number 13 / 427,040 (U.S. Patent No. 8,440,431); U.S. Patent Serial Number 13 / 427,137 (U.S. Patent No. 8,440,432); and U.S. Patent Serial Number 13 / 738,381, all of which are hereby incorporated by reference in their entirety.

[0161] As used herein, the terms "transfected", or "transformed", or "transduced" refer to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with an exogenous nucleic acid. Cells include primary target cells and their progeny.

[0162] A "transgene" is a gene or genetic material transferred from one organism to another. When a transgene is transferred into an organism, the organism can be called a transgenic organism. Generally, this term describes a segment of DNA containing a gene sequence that is isolated from one organism and introduced into a different organism. This non-natural segment of DNA can retain the ability to produce RNA or protein in the transgenic organism, or it can alter the normal function of the transgenic organism's genetic code. Generally, DNA is integrated into the germline of the organism. For example, in higher vertebrates, this can be achieved by injecting foreign DNA into the nucleus of a fertilized egg, or by somatic cell nuclear transfer where somatic cells in which the desired transgene(s) are integrated into the host genome are transferred into enucleated oocytes and transplanted into a surrogate mother to produce live offspring. A transgene can be a cDNA (complementary DNA) segment that is a copy of mRNA (messenger RNA) when inserted into a cell, or the gene itself that exists in the original region of genomic DNA. A transgene can be a genomic sequence, especially when introduced as a large clone into a BAC (bacterial artificial chromosome) or cosmid, or it can be in the form of a "mini-gene" often characterized by a combination of genomic DNA (including intron regions, e.g., intron 1), 5' or 3' regulatory regions together with a cDNA region. In the context of this specification, "expression" of a transgene, unless otherwise specified, means that a peptide sequence from a non-natural nucleic acid is expressed in at least one cell in the host. The peptide can be expressed from a transgene integrated into the host genome. A transgene can contain a polynucleotide encoding a protein or a fragment thereof (e.g., a functional fragment). A fragment of a protein (e.g., a functional fragment) can contain at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the amino acid sequence of the protein. A fragment of a protein can be a functional fragment of the protein. A functional fragment of a protein can retain part or all of the function of the protein.

[0163] As used herein, the term "transplantation tolerance" is defined as a state of donor-specific unresponsiveness that does not require continued pharmacological immunosuppression. Transplantation tolerance may eliminate many of the adverse events associated with immunosuppressive agents. Thus, induction of tolerance can result in improved acceptance of xenografts. In one embodiment, induction of tolerance can be identified by a reduction in the clinical symptoms of xenograft rejection. In another embodiment, induction of tolerance can improve or prevent metabolic, inflammatory and proliferative pathological conditions or diseases associated with xenograft transplantation. In yet another embodiment, induction of tolerance can improve or reduce or prevent adverse clinical conditions or diseases associated with the administration of immunosuppressive therapy used to prevent xenograft rejection. In still yet another embodiment, induction of tolerance can promote xenograft survival. In different embodiments, induction of tolerance can prevent recurrence in patients exhibiting these diseases or conditions.

[0164] The term "ungulate" refers to hoofed mammals. Artiodactyls are hoofed mammals with an even number of toes (cloven hooves) that include antelopes, camels, cows, deer, goats, pigs and sheep. Perissodactyls are hoofed mammals with an odd number of toes and include horses, zebras, rhinos and tapirs. The term ungulate, as used herein, refers to adult, embryonic or fetal ungulate animals.

[0165] As used herein, the term "vector" refers to a portion capable of transferring a polynucleotide into a host cell. Vectors include nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that contain one or more free ends or do not contain free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other types of polynucleotides known in the art, but are not limited thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which DNA or RNA sequences derived from a virus are present in the vector for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of self-replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell and are thereby replicated with the host genome. Further, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Common expression vectors useful in recombinant DNA technology are often in the form of plasmids. A recombinant expression vector can contain the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector contains one or more regulatory elements that can be selected based on the host cell used for expression and are operably linked to the nucleic acid sequence to be expressed.In recombinant expression vectors, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that enables expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or, when the vector is introduced into a host cell, in the host cell). For recombinant and cloning methods, reference is made to U.S. Patent Application Serial Number 10 / 815,730, the contents of which are hereby incorporated by reference in their entirety. Preferably, the vector is a DNA vector, more preferably capable of expressing RNA encoding the protein according to the invention. A number of suitable vectors are documented in the art; examples can be found in Molecular Cloning: a Laboratory Manual: 2nd Edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press or DNA cloning: a practical approach, Volume II: Expression systems, edited by D.M. Glover (IRL Press, 1995). can be.

[0166] As used herein, the term "zinc finger nuclease" or "ZFN" refers to an engineered (manipulated) DNA-binding protein that contains a zinc finger DNA-binding domain and a DNA cleavage domain. The zinc finger domain can be engineered to target a specific desired DNA sequence, which enables the zinc finger nuclease to target a unique sequence within the complex genome. They facilitate targeted editing of the genome by generating a double-strand break in the DNA at a user-specified location. Each ZFN contains two functional domains: a) a DNA-binding domain composed of strands of two-finger modules, each of which recognizes a specific hexamer (6 bp) sequence of DNA. The two-finger modules are stitched together to form a zinc finger protein with a specificity of at least 24 bp each. b) A DNA cleavage domain composed of the nuclease domain of Fok I. When the DNA-binding and DNA cleavage domains are fused, a highly specific pair of "genomic scissors" is created. ZFNs are gene editing tools.

[0167] A. Transgenic animals The present invention provides transgenic animals (e.g., transgenic pig animals) that serve as a source of organs, organ fragments, tissues or cells for use in xenotransplantation. The present invention extends to organs, tissues and cells derived from transgenic animals, and to groups of such animals, such as production herds.

[0168] The animal can be any suitable animal. In an exemplary embodiment, the animal is an ungulate, and more particularly, a pig animal or a pig.

[0169] The transgenic donor animal (e.g., an ungulate, a porcine animal or a pig) is genetically modified, and more particularly, contains multiple transgenes, for example, multiple transgenes at a single locus. In certain embodiments, the transgenic donor animal is genetically modified to express multiple transgenes that are split between a first locus (i.e., locus 1) and a second locus (i.e., locus 2). The locus may be a natural or a modified natural locus. Various strategies for modifying natural loci to facilitate targeting are described herein.

[0170] In an exemplary embodiment, the invention provides a transgenic animal (e.g., a transgenic porcine animal) comprising the integration and expression of at least four transgenes at a single locus under the control of at least two promoters (e.g., an exogenous promoter, or a combination of an exogenous and a natural promoter), wherein the pig lacks expression of alpha1,3-galactosyltransferase. Optionally, the transgenic animal comprises one or more additional genetic modifications including, without limitation, addition and / or deletion of genes including knockouts and knockins, as well as gene replacement and rearrangement.

[0171] In certain embodiments, the present invention provides a transgenic porcine animal comprising at least four transgenes integrated and expressed at a single locus, wherein the expression of the at least four transgenes is controlled by dedicated promoters, i.e., the promoters drive the expression of each individual transgene. For example, when a transgenic animal integrates and expresses four transgenes at a single locus, the expression of those transgenes is driven by four promoters, each promoter being specific for a particular transgene. In alternative embodiments, a given promoter controls the expression of more than one transgene (e.g., two transgenes, three transgenes). For example, when a transgenic animal integrates and expresses four transgenes, two of the four transgenes are expressed as a polycistron controlled by a first promoter and two of the four transgenes are expressed as a polycistron controlled by a second promoter.

[0172] In exemplary embodiments, the at least four transgenes are selected from the group consisting of immunomodulatory factors (e.g., immunosuppressive factors), anticoagulation factors, complement inhibitory factors, and cryoprotective factor transgenes.

[0173] In exemplary embodiments, the single locus is a natural locus. In other embodiments, the single locus is a modified natural locus, e.g., a transgenic locus. The transgenic locus may be, for example, a locus containing a selectable marker gene or a locus containing a landing pad.

[0174] In exemplary embodiments, the at least four transgenes are provided in a multicistronic vector (MCV) and are integrated by random integration or by using gene editing tools. Optionally, the transgenic animal can have one or more additional genetic modifications. The additional genetic modification can be, for example, a gene knockout or a gene knock-in. In certain embodiments, the additional genetic modification comprises a chimeric porcine-human vWF.

[0175] In another embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising (i) a lack of expression of alpha1, galactosyltransferase (i.e., alphaGal null), and (ii) at least five genetic modifications resulting in the integration and expression of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 transgenes at a single locus. The expression of the transgenes is driven by a dedicated promoter or a promoter that controls the expression of two or more transgenes. The promoter may be exogenous or a combination of exogenous and native promoters.

[0176] In certain embodiments, to better modulate the expression of the transgene combination, more than four additional transgenes may involve the integration of transgenes at more than one locus (e.g., the integration of at least 4 transgenes under the control of at least 2 promoters integrated into GGTA1, and a second polycistronic integration at a second locus (e.g., CMAH or B4GalNT2 or AAVS1 or Rosa26)). In certain embodiments where the second locus is genetically modified, such a second locus may be modified to inactivate the expression of another porcine gene (e.g., through the application of gene editing and / or homologous recombination techniques). In an exemplary embodiment, the plurality of transgenes integrated and expressed as the second locus are selected from the group consisting of immunomodulatory factor, complement inhibitory factor, anticoagulant factor, and cryoprotective factor transgenes. In an exemplary embodiment, the second locus is a native locus, a modified native locus, or a transgenic locus (e.g., a landing pad). In an exemplary embodiment, at least two transgenes of the second locus are provided by MCV and integrated using gene editing tools. Optionally, the transgenic animal can have one or more additional genetic modifications.

[0177] In one embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising at least four genetic modifications that result in (i) reduced expression of alpha1, galactosyltransferase, and (ii) integration and expression of at least four transgenes expressed under the control of at least two promoters (e.g., an exogenous promoter, or a combination of an exogenous and a native promoter) at a single locus. In an exemplary embodiment, the transgenes are selected from the group consisting of an immunomodulatory factor, an anticoagulant factor, a complement inhibitor factor, and a cryoprotective factor transgene. In an exemplary embodiment, the single locus is a native locus, a modified native locus, or a transgenic locus (e.g., a landing pad). In an exemplary embodiment, at least two of the transgenes are provided by an MCV and are integrated using a gene editing tool (i.e., CRISPR / cas9, TALEN, or ZFN) to enhance the efficiency of homologous recombination or homology-dependent repair. Optionally, the transgenic animal can have one or more additional genetic modifications.

[0178] In another embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising at least five genetic modifications that result in (i) reduced expression of alpha1, galactosyltransferase and (ii) integration and expression of at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten transgenes, either at a single locus or split between two loci. In an exemplary embodiment, the transgenes are selected from the group consisting of immunomodulatory factors, complement inhibitory factors, anticoagulant factors and cryoprotective factor transgenes. In an exemplary embodiment, the single locus is a natural locus, a modified natural locus or a transgenic locus (e.g., a landing pad). In an exemplary embodiment, at least two transgenes are provided by MCV and integrated using a gene editing tool (i.e., CRISPR / cas9, TALEN or ZFN) to enhance the efficiency of homologous recombination or homology-dependent repair. Optionally, the transgenic animal can have one or more additional genetic modifications.

[0179] In an exemplary embodiment, the transgenic animal lacks expression of alpha1, galactosyltransferase (i.e., alphaGal null) and comprises at least one, at least two, at least three, at least four, at least five, at least six or at least seven or more genetic modifications. Optionally, in addition to integration of the transgenes, further knockouts include knockout of the beta4GalNT2 gene or the CMAH gene (both genes associated with causes of innate immunity and rejection of xenografts).

[0180] In an exemplary embodiment, the transgenic animal has reduced expression of alpha1, galactosyltransferase and comprises at least one, at least two, at least three, at least four, at least five, at least six or at least seven additional genetic modifications.

[0181] In certain embodiments, the expression of alpha1,3-galactosyltransferase is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 95%.

[0182] In exemplary embodiments, the transgenic animal comprises (i) a genetic modification that results in a lack of expression of alpha1,3-galactosyltransferase, and (ii) at least four additional genetic modifications, or more specifically four additional genetic modifications. These additional genetic modifications may be any suitable genetic modifications including, but not limited to, gene replacements (INDELs) including CRISPR-induced deletions / insertions or knockouts or knockins at other loci (e.g., B4GalNT2, CMAH, vWF).

[0183] In exemplary embodiments, the transgenic animal comprises (i) a genetic modification that results in a reduction of the expression of alpha1,3-galactosyltransferase, and (ii) at least four additional genetic modifications, or more specifically four additional genetic modifications.

[0184] In exemplary embodiments, the transgenic animal comprises (i) a genetic modification that results in a lack of expression of alpha1,3-galactosyltransferase, and (ii) at least five additional genetic modifications, or more specifically five additional genetic modifications.

[0185] In exemplary embodiments, the transgenic animal comprises (i) a genetic modification that results in a reduction of the expression of alpha1,3-galactosyltransferase, and (ii) at least five additional genetic modifications, or more specifically at least five additional genetic modifications.

[0186] In exemplary embodiments, the transgenic animal comprises (i) a genetic modification that results in a lack of expression of alpha1,3-galactosyltransferase, and (ii) at least six additional genetic modifications, or more specifically six additional genetic modifications.

[0187] In an exemplary embodiment, the transgenic animal comprises (i) a genetic modification that results in reduced expression of alpha 1,3 galactosyltransferase, and (ii) at least six additional genetic modifications, or more particularly six additional genetic modifications.

[0188] In certain embodiments, the donor animal (e.g., an ungulate, a porcine animal or a pig) comprises (i) a lack of expression of alpha 1,3 galactosyltransferase, and (ii) a genetic modification that results in the integration and expression of at least one, at least two, at least three, at least four, at least five or at least six or more transgenes.

[0189] In an exemplary embodiment, the invention provides a porcine animal comprising (i) a lack of expression of alpha 1,3 galactosyltransferase, and (ii) a genetic modification that results in the integration and expression of at least four additional transgenes.

[0190] In an exemplary embodiment, the invention provides a porcine animal comprising (i) a lack of expression of alpha 1,3 galactosyltransferase, and (ii) a genetic modification that results in the integration and expression of at least five additional transgenes, or more particularly five additional genetic modifications.

[0191] In an exemplary embodiment, the invention provides a porcine animal comprising (i) a lack of expression of alpha 1,3 galactosyltransferase, and (ii) a genetic modification that results in the integration and expression of at least six additional transgenes, or more particularly six additional genetic modifications.

[0192] In certain embodiments, the donor animal (e.g., ungulate, porcine animal or pig) comprises a genetic modification that results in (i) reduced expression of alpha1,3 galactosyltransferase, and (ii) the incorporation and expression of at least 4, at least 5, or at least 6, or more transgenes, or more specifically 4, 5 or at least 6 additional transgenes.

[0193] In an exemplary embodiment, the donor animal (e.g., ungulate, porcine animal or pig) comprises a genetic modification that results in (i) reduced expression of alpha1,3 galactosyltransferase, and (ii) the incorporation and expression of 5 additional transgenes. Optionally, the donor animal can contain one or more additional genetic modifications.

[0194] In an exemplary embodiment, the donor animal (e.g., ungulate, porcine animal or pig) comprises a genetic modification that results in (i) reduced expression of alpha1,3 galactosyltransferase, and (ii) the incorporation and expression of 6 additional transgenes. Optionally, the donor animal can contain one or more additional genetic modifications (knockout, knockin, INDEL, modification of porcine vWF).

[0195] B. Transgene Expression Expression of the transgene can be at any level, but in specific embodiments, the expression is at a high level.

[0196] Depending on the desired level and tissue-specific expression, various promoter / enhancer elements can be used. Depending on the desired expression pattern, the promoter / enhancer can be constitutive or inducible. The promoter can be exogenous or native, or a combination of exogenous and native promoters.

[0197] In certain embodiments, the transgene is expressed from a constitutive or ubiquitous promoter. In certain other embodiments, the transgene is expressed from a tissue-specific or cell-type specific promoter, or an inducible promoter, and can include additional regulatory elements such as enhancers, insulators, matrix attachment regions (MARs).

[0198] In exemplary embodiments, four or more transgenes are co-expressed. In exemplary embodiments, four or more transgenes are expressed at approximately equimolar amounts.

[0199] In exemplary embodiments, the transgene is expressed by a promoter that is primarily active in endothelial cells. In certain embodiments, the expression of the transgene is controlled by the porcine Icam-2 enhancer / promoter.

[0200] In certain embodiments, the expression of the transgene is controlled by a constitutive CAG promoter.

[0201] In certain embodiments, the transgenic animal is genetically modified to effect the integration and expression of two or more transgenes, wherein at least one transgene is controlled by a constitutive promoter and at least one transgene is controlled by a tissue-specific promoter, or more particularly, a promoter that is primarily active in endothelial cells.

[0202] In exemplary embodiments, the transgenic animal is genetically modified to effect the integration and expression of four or more transgenes at a single locus, wherein at least one transgene is controlled by a constitutive promoter and at least one transgene is controlled by a tissue-specific promoter, or more particularly, a promoter that is primarily active in endothelial cells.

[0203] The transgene can be any transgene suitable for use in modifying a donor animal (e.g., a porcine animal) for use in xenotransplantation. In an exemplary embodiment, the transgene is selected from immunomodulatory factors (e.g., complement regulatory factors, complement inhibitory factors, immunosuppressive factors), anticoagulant factors, cryoprotective factor genes, or combinations thereof. In certain embodiments, the sequence of the transgene is human.

[0204] In certain embodiments, the transgene is an immunomodulatory factor.

[0205] In certain embodiments, the transgene is a complement regulatory factor, or more specifically, a complement inhibitory factor. Complement inhibitory factors can include, without limitation, CD46 (MCP), CD59, or CR1. The sequence of the complement inhibitory factor can be human.

[0206] In certain embodiments, the transgene is a complement pathway inhibitor (i.e., a complement inhibitory factor). Complement inhibitory factors can include, without limitation, CD55, CD59, CR1, and CD46 (MCP). The sequence of the complement inhibitory factor can be human.

[0207] In certain embodiments, the transgene is an immunosuppressive factor.

[0208] The complement inhibitory factor can be human CD46 (hCD46), and expression can be through a minigene construct (see Loveland et al., Xenotransplantation, Vol. 11(2):171 - 183, 20 04).

[0209] In certain embodiments, the transgene is an immunosuppressive gene having the effect of modulating T cells, such as CTLA4-Ig, or a dominant negative inhibitor of class II MHC (CIITA), or other genes that modulate the expression of B cell or T cell-mediated immune functions. In further embodiments, such animals may be further modified to eliminate the expression of genes that affect immune function. In certain embodiments, the immunosuppressive factor is CD47.

[0210] In certain embodiments, the transgene is an anticoagulant factor. Anticoagulant factors can include, without limitation, tissue factor pathway inhibitor (TFPI), hirudin, thrombomodulin (TBM), endothelial protein C receptor (EPCR), and CD39. The sequence of the anticoagulant factor may be human.

[0211] The transgenic animal can also have one or more additional genetic modifications.

[0212] In one embodiment, the animal can be genetically modified to inhibit the expression of the CMP-Neu5Ac hydroxylase gene (CMAH) (see, for example, U.S. Patent Publication 2005-0223418), the iGb3 synthase gene (see, for example, U.S. Patent Publication 2005-0155095), and / or the Forssman synthase gene (see, for example, U.S. Patent Publication 2006-0068479). Further, the animal can be genetically modified to reduce the expression of procoagulant factors. In particular, in one embodiment, the animal is genetically modified to reduce or eliminate the expression of procoagulant genes such as FGL2 (fibrinogen-like protein 2) (see, for example, Marsden et al. (2003) J din Invest. 112:58-66; Ghanekar et al. (2004) J. Immunol. 172:5693-701; Mendicino et al. (2005) Circulation. 112:248-56; Mu et al. (20 07) Physiol Genomics. 31(1):53-62).

[0213] In another embodiment, the animal can be genetically modified to inhibit the expression of beta-1,4 N-acetylgalactosaminyltransferase 2 (β4GalNT2).

[0214] C. Specific Genetics 1. Alpha1,3 galactosyltransferase (alpha-Gal) In one embodiment, the present invention provides a transgenic animal suitable for use as a source of organs, tissues and cells for xenotransplantation, wherein the donor animal lacks or has reduced expression of alpha-Gal. Transgenic animals lacking expression of alpha-Gal (i.e., alpha-Gal null) have one or more additional genetic modifications, in certain embodiments at least 4 additional genetic modifications, at least 5 additional genetic modifications or at least 6 additional genetic modifications. These genetic modifications can be, for example, the integration or expression of transgenes. In certain embodiments, the transgenic animal has (i) a lack of expression of alpha-Gal; and (ii) at least 3 genetic modifications resulting in the integration and expression of at least 2 transgenes at a single locus. In certain embodiments, the single locus is a modified alpha-Gal.

[0215] To eliminate or modulate the anti-Gal humoral response caused by xenotransplantation, enzymatic removal of epitopes by alpha-galactosidase (Stone et al., Transplantation, 63:640-645 (1997)), specific anti-gal antibody removal (Ye et al., Transplantation, 58:330-337, 1994), alphaGT expression could not be eliminated Epitope capping with other hydrocarbon moieties (Tanemura et al., J. Biol. Chem. 273, 16421-16425 (1998) and Koike et al., Xenotransplantation, 4, 147-153 (1997)), and introduction of complement inhibitory proteins (Dalmasso et al., Clin. Exp. Immunol. 86, 31-35 (1991), Dalmasso et al., Transplantation, 52, 530-533 (1991)) have been implemented in various strategies. Competitive inhibition of αGT in transgenic pigs has been reported by C. Costa et al. (FASEB J, 13, 1762 (1999)) to result in only a partial reduction in the number of epitopes. Similarly, attempts to block the expression of gal epitopes in N-acetylglucosaminyltransferase III transgenic pigs have also been reported by S. Miyagawa et al. (J. Biol. Chem. 276, 39310 (2000)) to result in only a partial reduction in the number of gal epitopes and to be unable to significantly prolong graft survival in primate recipients. A single allele knockout of the alphaGal locus in porcine cells and live animals has been reported. Denning et al. (Nature Biotechnology, 19, 559-562 (2001)) reported targeted gene deletion of one allele of the αGT gene in sheep. Harrison et al. (Transgenics Research, 11, 143-

[0216] ​(Science, 295:1089-1092, 2002) reported the production of heterozygous αGT knockout somatic porcine fetal fibroblasts in 2002. In 2002, Lai et al. (Science, 295:1089-1092, 2002) and Dai et al. (Nature Biotechnology, 20:251-255, 2002) reported the production of pigs in which one allele of the αGT gene was successfully inactivated. Also, when the inactivation of alpha Gal was through targeted insertion of the marker gene neomycin phosphotransferase (Neo) that interfered with the coding region of the alpha Gal gene (Ramsoondar et al. (Biol of Reproduc, 69:437-445 (2003)), the production of heterozygous αGT knockout pigs that expressed both HT and the alpha Gal epitope and also expressed human alpha-1,2-fucosyltransferase (HT) was reported. PCT Publication No. WO03 / 055302 to the University of Missouri administrators confirmed the production of heterozygous alpha Gal knockout miniature pigs for use in xenotransplantation, in which the expression of functional αGT in the knockout pigs was reduced compared to the wild type.

[0217] PCT Publication No. WO94 / 21799 and U.S. Patent No. 5,821,117 to the Austin Research Institute; PCT Publication No. WO95 / 20661 to Bresatec; and PCT Publication No. WO95 / 28412, U.S. Patent No. 6,153,428, U.S. Patent No. 6,413,769, and U.S. Publication No. 2003 / 0014770 to BioTransplant, Inc. and The General Hospital Corporation provide discussions on the production of αGT-negative porcine cells based on the cDNA of the αGT gene. A major breakthrough in the field of xenotransplantation was the production of the first live pigs lacking any functional expression of alpha Gal (Phelps et al., Science, 299:411-414 (2003); Revivico (see also PCT Publication No. WO04 / 028243 by r, Inc. and PCT Publication No. WO04 / 016742 by Immerge Biotherapeutics, Inc.).

[0218] In one embodiment, the animal (and organs, tissues, and cells derived therefrom) is provided from a transgenic animal (e.g., a transgenic pig) that contains at least four transgenes, where the four transgenes are integrated and expressed at a single locus under the control of at least two promoters, and the pig lacks expression of alpha1,3 galactosyltransferase. In an exemplary embodiment, the transgenes are integrated and expressed at a modified alphaGal locus. In certain embodiments, the at least two promoters are exogenous, native, or a combination of exogenous and native.

[0219] In one embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that (i) lacks any expression of functional alphaGal and (ii) integrates and expresses at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more transgenes at a single locus. In an exemplary embodiment, the transgenes are integrated and expressed at a modified alphaGal locus.

[0220] In certain embodiments, the animal can include one or more additional genetic modifications. These genetic modifications can result in the integration and expression of one or more additional transgenes at the same locus or at different loci.

[0221] In one embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks any expression of functional alphaGal and integrates and expresses at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 additional transgenes.

[0222] In another embodiment, animals, organs, tissues, and cells are provided that have a reduced level of functional alphaGal expression and incorporate and express at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 additional transgenes. The expression of functional alphaGal can be reduced, for example, by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.

[0223] The absence or reduced level of functional αGT expression can be achieved by any suitable means. In an embodiment, an animal (e.g., an ungulate, a porcine animal) is provided in which one allele of the alphaGal gene is inactivated through a gene targeting event. In another embodiment, a porcine animal is provided in which both alleles of the alphaGal gene are inactivated through a gene targeting event. In one embodiment, the gene can be targeted through homologous recombination. In other embodiments, the gene can be disrupted, i.e., a portion of the gene code can be altered, thereby affecting the transcription and / or translation of that segment of the gene. For example, gene disruption can occur by substitution, deletion ("knockout"), or insertion ("knockin") techniques that include the targeted insertion of a selectable marker gene (e.g., neo) that interferes with the coding region of the alphaGal gene. Additional genes for desired proteins or regulatory sequences that modulate the transcription of existing sequences can be inserted.

[0224] In certain embodiments, alleles of the alphaGal gene are inactivated such that the resulting alphaGal enzyme can no longer produce Gal at the cell surface. In one embodiment, the alphaGal gene can be transcribed into RNA but not translated into protein. In another embodiment, the alphaGal gene can be transcribed in a truncated form. Such truncated RNA may or may not be translated into a non-functional protein. In alternative embodiments, the alphaGal gene can be inactivated such that transcription of the gene does not occur. In further embodiments, the alphaGal gene can be transcribed and then translated into a non-functional protein.

[0225] In some embodiments, expression of the active alphaGal gene can be reduced by the use of alternative methods, such as methods targeting gene transcription or translation. For example, expression can be reduced by the use of antisense RNA or siRNA targeting the native αGT gene or its mRNA. In other embodiments, site-specific recombinases are used to target genomic regions for recombination. Examples of such systems are the CRE-lox system and the Flp-Frt system.

[0226] Pigs having two inactive alleles of the alphaGal gene do not occur naturally. It has previously been found that point mutations were identified that blocked the second allele of the alphaGal gene from producing a functional alphaGal enzyme while attempting to knockout the second allele of the alphaGal gene through gene targeting events.

[0227] Accordingly, in another aspect of the present invention, alphaGal can be inactivated through at least one point mutation. In one embodiment, one allele of the alphaGal gene can be inactivated through at least one point mutation. In another embodiment, both alleles of the alphaGal gene can be inactivated through at least one point mutation. In one embodiment, this point mutation can occur through a gene targeting event. In another embodiment, this point mutation can be naturally occurring. In a further embodiment, a mutagen can be used to induce a mutation in the alphaGal gene.

[0228] 2.β4GaINT2 In one embodiment, the present invention provides a transgenic animal suitable for use as a source of organs, tissues, and cells for xenotransplantation, wherein the donor animal lacks or has reduced expression of beta1,4N-acetyl-galactosaminyltransferase 2 (β4GALNT2). A transgenic animal lacking (i.e., β4GALNT2 null) expression of β4GALNT2 has one or more additional genetic modifications. These genetic modifications can be, for example, the integration or expression of a transgene. In certain embodiments, a transgenic animal lacking or having reduced expression of beta1,4N-acetyl-galactosaminyltransferase 2 (β4GALNT2) is also characterized by (i) lack of expression of alphaGal; and (ii) integration and expression of at least four transgenes at a single locus under the control of at least two promoters.

[0229] The glycans produced by β4Gal-NT2 are xenogenic antigens for many humans. Estrada JL et al., Xenotransplantation, 2015;22:194-202. In humans and mice, β4GALNT2 catalyzes the addition of N-acetylgalactosamine to sialic acid-modified lactosamine to produce GalNAcb1-4(Neu5Ac a2-3)Galb1-4GlcNAc b1-3Gal, the Sda blood group antigen. This gene is functional in porcine transplantable organs (kidney, heart, liver, lung and pancreas) and endothelial cells. Approximately 5% of humans have inactive β4GalNT2, and as a result, generate antibodies against the Sda and CAD carbohydrates produced by this gene. See Byrne GW et al., Transplantation, 2011;91:287-292; Byrne GW et al., Xenotransplantation, 2014;21:543-554.

[0230] Any suitable method can be used to generate pigs whose genome lacks or has reduced expression of endogenous β4GALNT2. Disruptions can be placed at many sites in the endogenous porcine β4GALNT2 nucleic acid sequence. Examples of disruptions include, but are not limited to, deletions in the native gene sequence and insertions of heterologous nucleic acid sequences into the native gene sequence. Examples of insertions can include, but are not limited to, an artificial splice acceptor linked to a stop codon or a splice donor linked to a fusion partner such as GFP. The knockout construct can contain a sequence that is homologous to the endogenous β4GALNT2 nucleic acid sequence or to a sequence adjacent to the endogenous β4GALNT2 nucleic acid sequence. In some cases, the knockout construct can contain a nucleic acid sequence encoding a selectable marker (e.g., antibiotic resistance, a fluorescent reporter (e.g., GFP or YFP), or an enzyme (e.g., β-galactosidase)) operably linked to a regulatory sequence (e.g., a promoter). The knockout construct can contain other nucleic acid sequences, such as recombination sequences (e.g., loxP sequences, Sendai et al., Transplantation, 81(5):760- (see page 766 (2006)), and can include splice acceptor sequences, splice donor sequences, transcription start sequences, and transcription termination sequences. Disruptions in the endogenous β4GALNT2 nucleic acid sequence can result in reduced gene expression, or non-functional truncation or fusion of the encoded polypeptide.

[0231] In one embodiment, the invention provides a transgenic animal (e.g., a porcine animal) that expresses reduced or no β4GALNT2. Optionally, the animal includes one or more additional genetic modifications.

[0232] In an exemplary embodiment, the invention provides a transgenic animal (e.g., a porcine animal) that incorporates and expresses at least four transgenes under the control of at least two promoters, wherein the animal lacks or has reduced expression of β4GALNT2. Optionally, the animal includes one or more additional genetic modifications.

[0233] In one embodiment, the invention provides a transgenic animal (e.g., a porcine animal) that expresses reduced or no Sda or Sda-like glycan produced by porcine β4GALNT2. Optionally, the animal includes one or more additional genetic modifications.

[0234] In an exemplary embodiment, the invention provides a transgenic animal (e.g., a porcine animal) that incorporates and expresses at least four transgenes under the control of at least two promoters, and the animal lacks or has reduced expression of Sda or Sda-like glycan produced from porcine β4GALNT2. Optionally, the animal includes one or more additional genetic modifications.

[0235] 3. CMAH In one embodiment, the present invention provides a transgenic animal suitable for use as a source of organs, tissues and cells for xenotransplantation, wherein the donor animal lacks or has reduced expression of cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). The transgenic animal lacking CMAH expression (CMAH null) has one or more additional genetic modifications. These genetic modifications may be, for example, the integration or expression of a transgene. In certain embodiments, the transgenic animal has (i) a lack of expression of alpha Gal; and (ii) at least four additional genetic modifications that result in the integration and expression of at least four transgenes at a single locus.

[0236] Porcine cells express cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), which is not found in human cells. CMAH converts sialic acid N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Thus, when porcine tissue is transplanted into a human, this epitope elicits an antibody-mediated rejection reaction from the human patient immediately after transplantation. See Varki A., Am J Phys Anthropol, 200 1 year; (Suppl 33):54-69; Zhu A., Xenotransplantation, 2002; 9:376-381; Miwa Y., Xenotransplantation, 2004; 11:247- 253; Tahara H., J Immunol, 2010; 184:3269-3275.

[0237] Any suitable method can be used to generate pigs whose genome lacks or has reduced expression of endogenous CMAH. Disruptions can be placed at many sites in the endogenous porcine CMAH nucleic acid sequence. Examples of disruptions include, but are not limited to, deletions in the native gene sequence and insertions of heterologous nucleic acid sequences into the native gene sequence. Examples of insertions can include, but are not limited to, an artificial splice acceptor linked to a stop codon, or a splice donor linked to a fusion partner such as GFP. The knockout construct can contain a sequence that is homologous to the endogenous CMAH nucleic acid sequence or to a sequence adjacent to the endogenous CMAH nucleic acid sequence. In some cases, the knockout construct can contain a nucleic acid sequence encoding a selectable marker (e.g., antibiotic resistance, a fluorescent reporter (e.g., GFP or YFP), or an enzyme (e.g., β-galactosidase)) operably linked to a regulatory sequence (e.g., a promoter). The knockout construct can contain other nucleic acid sequences, such as recombinant sequences (e.g., loxP sequences, see Sendai et al., Transplantation, Vol. 81(5):760 - 766 (2006)), and can include splice acceptor sequences, splice donor sequences, transcription start sequences, and transcription termination sequences. Disruptions in the endogenous CMAH nucleic acid sequence can result in reduced gene expression or non-functional truncation or fusion of the encoded polypeptide.

[0238] In one embodiment, the invention provides transgenic animals (e.g., porcine animals) that express reduced or no CMAH glycosyltransferase. Optionally, the animals can contain one or more additional genetic modifications.

[0239] In an exemplary embodiment, the present invention provides a transgenic animal (e.g., a porcine animal) that incorporates and expresses at least four transgenes under the control of at least two promoters, wherein the animal lacks or has reduced expression of CMAH. Optionally, the animal includes one or more additional genetic modifications.

[0240] 4.vWF The von Willebrand factor (vWF) gene is a large complex gene that has multiple domains and encodes a multimeric glycoprotein. (Ulrichts, H, Udvardy M, Lenting PJ, Pareyn I et al., Shielding of the A1 domain by the D'D3 domains of von Willebrand Factor Modulates Its interaction with Platelet Glycoprotein 1b-IX-V. (2006) JBC, 281:4699-4707; Zhou Y-F, Eng ET 、Zhu J, Lu C et al., Sequence and structure relationships within von Willebrand factor. (2012) Blood, 120:449-458). The main function of the multimeric glycoprotein von Willebrand factor (vWF) is platelet adhesion to connective tissue and the subendothelium and platelet aggregation in response to vWF binding to platelet glycoprotein Ib (GPIb). However, this phenomenon is more disadvantageous during xenotransplantation when recipient platelet aggregation has a detrimental effect on the survival of the transplanted organ. As an example, transplantation of porcine lungs (and other organs) into humans or non-human primates results in spontaneous aggregation and sequestration of human platelets. This can be avoided by "humanizing" the porcine VWF gene in an effort to eliminate this spontaneous binding of porcine vWF to human platelets.

[0241] Generally, humanization or modification of the porcine vWF gene requires deletion of the gene sequence(s) associated with spontaneous aggregation of human platelets and replacement with the human gene counterpart that does not generate spontaneous aggregation. This can include deletion of all or part of the porcine vWF gene with replacement with all or part of the human vWF gene.

[0242] Modification of porcine vWF aimed at elimination of the spontaneous platelet aggregation response can include regions within the D3 (partial), A1, A2, A3 (partial) domains, which are known to be associated with folding and sequestration of the GP1b binding site (D3 domain) of hvWF, as well as regions associated with the GP1b receptor (A1 domain) and the ADAMTS13 cleavage site (A2 domain). Exons 22-28 encompass these regions. Human platelets spontaneously aggregate in the presence of porcine blood under normal stress forces. To avoid this potential threat for the success of xenotransplantation, and since human vWF does not induce spontaneous platelet aggregation under conditions of normal shear stress in blood, the regions of the human vWF gene associated with folding of the vWF protein as well as regions associated with GPib binding, collagen binding (one of the two regions), and ADAMTS13 cleavage may be utilized for exchange of the genomic homolog in the porcine vWF gene (and the resulting chimeric human / porcine protein). In this way, an alternative folding that hides or masks the GP1b binding site on vWF as well as the humanized receptor sites in the A domains may be provided by a single cDNA or genomic fragment from the human vWF gene. This may be achievable through homologous recombination or gene targeting, including cases where such mechanisms are enhanced using gene editing methods. (For example, CRISPR-assisted homologous recombination can be used to incorporate a human vWF fragment into the porcine vWF locus. This human fragment may exchange the regions linked to spontaneous platelet aggregation mentioned above and may be in the form of a cDNA or genomic fragment from the human vWF gene).

[0243] In an exemplary embodiment, the insertion of the relevant human vWF gene sequence can be carried out by any current method used for genome editing, for example, but not limited to, CRISPR / CAS9, TALEN nuclease. The modification of porcine vWF can be carried out by replacing only the relevant region of the porcine vWF gene or, alternatively, by replacing the entire pvWF gene with hvWF.

[0244] In one embodiment, the region of the porcine vWF gene can be replaced with the human counterpart (E22 - E28 region). Alternatively, the transgenic animal can have a complete knockout of the vWF gene and a complete exchange of the gene synthesis sequence of the human vWVF gene using a site - specific recombination system (i.e., the CRE - LOX recombination system and / or specific nucleobase pair modifications that replace nucleotides in the porcine vWF genomic sequence with human counterparts).

[0245] In one embodiment, the invention is a transgenic animal (e.g., a transgenic porcine animal) lacking the expression of alpha Gal and genetic modification to the porcine vWF gene. The modification can be, for example, a knockout of the porcine vWF gene and replacement with a humanized or chimeric vWF gene. The transgenic animal can contain one or more additional genetic modifications. In one embodiment, the transgenic animal further includes the integration and expression of CD46.

[0246] The transgenic animal can also be mated with a second transgenic animal containing one or more genetic modifications. For example, the invention is a transgenic animal (e.g., a transgenic porcine animal) lacking the expression of alpha Gal and genetic modification to the porcine vWF gene, which can be mated with a second transgenic animal that provides an animal containing multiple genetic modifications, where the second transgenic animal contains at least four transgenes at a single locus or at least four transgenes at a single locus and at least two transgenes at a second locus.

[0247] In one embodiment, the present invention is a transgenic animal (e.g., a transgenic porcine animal) lacking the expression of alphaGal, as well as genetic modification to the porcine vWF gene (e.g., chimeric human-porcine vWF), and at least four genetic modifications at a single locus under the control of at least two promoters. The loci may be different. In an exemplary embodiment, the locus is a natural locus or a modified natural locus. The locus may be, for example, AAVS1, ROSA26, CMAH, β4GalNT2, and GGTA1. At least four transgenes can be integrated by homologous recombination or gene editing tools.

[0248] 5. Transgene The transgene introduced into the genome of the transgenic animal of the present invention may be any suitable transgene.

[0249] (i) Immunomodulatory factor In one embodiment, the transgene is an immunomodulatory factor. In an exemplary embodiment, the donor animal is genetically modified such that as a result (i) it lacks or has reduced expression of alphaGal, and (ii) at least four transgenes are integrated and expressed at a single locus, and at least one of the at least two transgenes is an immunomodulatory factor.

[0250] The immunomodulatory factor may be any suitable immunomodulatory factor. In an exemplary embodiment, the immunomodulatory factor is a complement regulatory factor (e.g., a complement inhibitory factor) or an immunosuppressive factor.

[0251] A. Complement regulatory factor In one embodiment, the present invention provides a transgenic animal (e.g., a porcine animal) suitable for use as a source of organs, tissues, and cells for xenotransplantation, wherein the donor animal is genetically modified to incorporate and express at least one complement regulatory factor, such as a complement inhibitor. In an exemplary embodiment, the donor animal is genetically modified such that as a result (i) it lacks or has reduced expression of alphaGal, and (ii) at least four transgenes are incorporated and expressed at a single locus, and at least one of the transgenes is a complement regulatory factor, or more specifically, a complement inhibitor.

[0252] Complement is a general term for a series of blood proteins and is a major effector mechanism of the immune system. Activation of complement and its deposition on target structures can lead to direct complement-mediated cell lysis or, indirectly, to cell or tissue destruction through the generation of potent modulators of inflammation and the recruitment and activation of immune effector cells. Complement activation products that mediate tissue injury are generated at various points in the complement pathway. Inappropriate complement activation on host tissues plays an important role in the pathogenesis of many autoimmune and inflammatory diseases and is also the cause of many disease states associated with, for example, cardio-pulmonary inflammation and allograft rejection after transplantation. Complement deposition on host cell membranes is blocked by complement inhibitory proteins expressed on the cell surface.

[0253] The complement system consists of a collection of approximately 30 proteins and is one of the major effector mechanisms of the immune system. The complement cascade is activated mainly through the classical (usually antibody-dependent) or alternative (usually antibody-independent) pathways. Activation via either pathway leads to the generation of the C3 convertase, the central enzyme complex of the cascade. The C3 convertase cleaves serum C3 into C3a and C3b, and the latter covalently binds to the activation site, leading to further generation of the C3 convertase (amplification loop). The activation product C3b (and further C4b, generated only through the classical pathway) and its breakdown products are important opsonins and are involved in cell-mediated lysis of target cells (by phagocytes and NK cells) and in promoting the transport and solubilization of immune complexes. The C3 / C4 activation products and their receptors on various cells of the immune system are also important in modulating the cellular immune response. The C3 convertase participates in the formation of the complex C5 convertase, which cleaves C5 to give C5a and C5b. C5a has potent pro-inflammatory and chemotactic properties and can mobilize and activate immune effector cells. The formation of C5b initiates the terminal complement pathway, leading to the sequential assembly of the complement proteins C6, C7, C8, and (C9)n, forming the membrane attack complex (MAC or C5b-9). The formation of the MAC in the target cell membrane can result in direct cell lysis but can also cause cell activation and the expression / release of various inflammatory modulators.

[0254] There are two broad classes of membrane complement inhibitors: inhibitors of the complement activation pathways (which inhibit C3 convertase formation) and inhibitors of the terminal complement pathway (which inhibit MAC formation). Membrane inhibitors of complement activation include complement receptor 1 (CR1), decay-accelerating factor (DAF or CD55), and membrane cofactor protein (MCP or CD46). All of them have a protein structure consisting of various numbers of repeating units of approximately 60-70 amino acids, named short consensus repeats (SCR), which is a common feature of C3 / C4 binding proteins. Rodent homologs of human complement activation inhibitors have been identified. The rodent protein Cr1 is a widely distributed complement activation inhibitor that functions similarly to both DAF and MCP. Rodents also express DAF and MCP, but Cr1 appears to be the most important functional regulator of complement activation in rodents. There is no homolog of Cr1 found in humans, but studies of Cr1 and its use in animal models are clinically important.

[0255] Control of the terminal complement pathway and MAC formation in the host cell membrane occurs mainly through the activity of CD59, a widely distributed 20kD glycoprotein attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. CD59 binds to C8 and C9 in the assembled MAC and prevents membrane insertion.

[0256] Host cells are protected from their own complement by membrane-bound complement regulatory proteins such as DAF, MCP, and CD59. When an organ is transplanted into another species, the recipient's natural antibodies bind to the endothelium of the donor organ and activate complement, thereby initiating a rapid rejection reaction. In contrast to human cells, porcine cells have previously been shown to be very sensitive to human complement, presumably because the complement regulatory proteins on the porcine cell surface are ineffective against human complement. When an organ is transplanted into another species, the recipient's natural antibodies bind to the endothelium of the donor organ and activate complement, thereby initiating a rapid rejection reaction. Several strategies, including removal of IgM natural antibodies and systemic complement depletion or inhibition using sCR1, heparin, or C1 inhibitor, have been shown to prevent or delay rejection.

[0257] An alternative approach to the problem of rejection is to express human membrane-bound complement regulatory molecules in transgenic pigs. Transgenic pigs expressing the decay-accelerating factor DAF (CD55), the membrane cofactor protein MCP (CD46), and the membrane inhibitor of reactive lysis MIRL (CD59) were generated. (See Klymium et al., Mol Reprod Dev (20 10) 77:209-221). These human inhibitors were shown to be abundantly expressed in porcine vascular endothelium. Ex vivo perfusion of hearts from control animals with human blood caused complement-mediated destruction of the organ within minutes, whereas hearts obtained from transgenic animals were refractory to complement and survived for several hours.

[0258] As outlined above, the rationale for expressing human complement regulatory proteins in porcine organs to "humanize" them is based on the assumption that endogenous porcine regulatory proteins are inefficient in inhibiting human complement and thus contribute little to organ survival in the context of xenotransplantation. (Cantarovich et al., Xenotransplantation, 9:25, 2002; Kirchhof et al., Xenotransplantation, 11(5):396, 2004; Tjernberg et al., Transplantation, April 27, 2008; 85(8):1193 - 9 ). Furthermore, soluble complement inhibitors can prevent complement - mediated lysis of islets in vitro (Bennet et al., Transplantation, 69(5):711, 2000) .

[0259] U.S. Patent No. 7,462,466 to Morgan et al. describes the isolation and characterization of several porcine analogs of human complement regulatory proteins (CRPs). Studies have shown that porcine organs expressing human complement regulatory protein molecules are resistant to complement injury not because they express human CRP molecules, but because they express greatly increased amounts of functional CRP molecules. Morgan et al. found that increased expression of porcine CRP may be equally effective as donor organs expressing human complement regulatory proteins in protecting donor organs from complement injury leading to hyperacute rejection.

[0260] CD46 has been characterized as a protein with regulatory properties that can protect host cells from complement-mediated attack activated through both classical and alternative pathways (Barilla-LaBarca, M. L. et al., J. Immunol. 168, 6298-6304 (2002)). Human CD46 (hCD46) can provide protection from complement lysis during inflammation and humoral rejection mediated by low levels of natural or induced anti-Gal or anti-non-Gal antibodies. As a result, more islands can be transplanted and better protected from subsequent rejection, thus reducing the need for immunosuppression.

[0261] In one embodiment of the present invention, there are provided animals (and organs, tissues, and cells derived therefrom) that lack the expression of functional alphaGal (or have reduced expression of alphaGal) and are genetically modified to incorporate and express at least 1, at least 2, at least 3, or at least 4 or more complement inhibitory factors. The expression of the complement inhibitory factors may be ubiquitous or under the control of a tissue-specific promoter.

[0262] In an exemplary embodiment, the complement inhibitory factor is a membrane complement inhibitory factor. The membrane complement inhibitory factor may be either an inhibitor of the complement activation pathway (inhibiting C3 convertase formation) or an inhibitor of the terminal complement pathway (inhibiting MAC formation). Membrane inhibitors of complement activation include complement receptor 1 (CR1), decay-accelerating factor (DAF or CD55), membrane cofactor protein (MCP or CD46), etc. Membrane inhibitors of the terminal complement pathway may include CD59, etc.

[0263] In an exemplary embodiment, the invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that includes (i) a lack of expression of alphaGal; and (ii) a genetic modification that results in the integration and expression of at least four transgenes at a single locus under the control of at least two promoters, wherein at least one of the at least two transgenes is a complement regulatory factor, more specifically a complement inhibitor, and even more specifically a membrane complement inhibitor. The single locus can be selected from a natural locus, a modified natural locus, or a transgenic locus. In an exemplary embodiment, the at least four transgenes are provided as an MCV, and the integration can be random integration or facilitated by a gene targeting tool. Optionally, the transgenic animal includes one or more additional genetic modifications, including but not limited to modifications of the native porcine vWF, B4GalNT2, CMAH, or Forssman genes.

[0264] In an exemplary embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that includes at least four transgenes, wherein the four transgenes are integrated and expressed at a single locus under the control of at least two promoters, the pig lacks expression of alpha1,3-galactosyltransferase, and the at least four transgenes include at least one complement regulatory factor, more specifically at least one complement inhibitor. Additional transgenes can be, for example, immunosuppressive factors, cytoprotective genes, or combinations thereof. The single locus can be selected from a natural locus, a modified natural locus, or a transgenic locus. In an exemplary embodiment, the at least four transgenes are provided as an MCV, and the integration is random or facilitated by a gene targeting tool. Optionally, the transgenic animal includes one or more additional genetic modifications.

[0265] In an exemplary embodiment, there is provided an animal (and organs, tissues, and cells derived therefrom) that is genetically modified to lack (or have reduced expression of) functional alphaGal and to incorporate and express at least four additional transgenes, wherein at least one of at least two of the at least four additional transgenes is a complement inhibitor, more particularly at least two membrane complement inhibitors.

[0266] In an exemplary embodiment, there is provided an animal (and organs, tissues, and cells derived therefrom) that is genetically modified to lack (or have reduced expression of) functional alphaGal and to (i) incorporate and express at least two complement inhibitors, more particularly at least two membrane complement inhibitors, and (ii) incorporate and express at least two additional transgenes selected from anticoagulation factors, immunosuppressive factors, cytoprotective genes, or combinations thereof.

[0267] In one embodiment, there is provided an animal (and organs, tissues, and cells derived therefrom) that is genetically modified to lack (or have reduced expression of) functional alphaGal and to (i) incorporate and express CD46 and CD55 and (ii) incorporate and express at least two additional transgenes. In certain embodiments, the additional transgenes are selected from anticoagulation factors, immunosuppressive factors, cytoprotective genes, or combinations thereof.

[0268] In certain embodiments, there is provided an animal (and organs, tissues, and cells derived therefrom) that is genetically modified to lack (or have reduced expression of) functional alphaGal and to incorporate and express at least four transgenes under the control of at least two promoters, wherein at least one of the transgenes is CD46 and the expression is controlled by an endogenous promoter.

[0269] In another embodiment, animals (and organs, tissues and cells derived therefrom) are provided that lack (or have reduced expression of) functional alphaGal and are genetically modified to incorporate and express (i) CD46 and CD55 and (i) at least three additional transgenes. In certain embodiments, the additional transgenes are selected from anticoagulation factors, immunosuppressive factors, cytoprotective genes or combinations thereof. In an exemplary embodiment, the at least three additional transgenes include at least two anticoagulation factors. In an exemplary embodiment, the at least three additional transgenes include at least two anticoagulation factors and an immunosuppressive factor.

[0270] In another embodiment, animals (and organs, tissues and cells derived therefrom) are provided that lack (or have reduced expression of) functional alphaGal and are genetically modified to incorporate and express (i) CD46 and CD55 and (i) at least four additional transgenes. In certain embodiments, the additional transgenes are selected from anticoagulation factors, immunosuppressive factors, cytoprotective genes or combinations thereof. In an exemplary embodiment, the at least four additional transgenes include at least two anticoagulation factors. In an exemplary embodiment, the at least four additional transgenes include at least two anticoagulation factors and an immunosuppressive factor. In an exemplary embodiment, the at least four additional transgenes include at least three anticoagulation factors.

[0271] In another embodiment, animals (and organs, tissues, and cells derived therefrom) are provided that lack (or have reduced expression of) functional alphaGal and are genetically modified to incorporate and express (i) CD46 and CD55, and (i) at least five additional transgenes. In certain embodiments, the additional transgenes are selected from anticoagulation factors, immunosuppressive factors, cytoprotective genes, or combinations thereof. In an exemplary embodiment, the at least five additional transgenes include at least two anticoagulation factors and at least one immunosuppressive factor. In an exemplary embodiment, the at least five additional transgenes include at least three anticoagulation factors and at least one immunosuppressive factor. In an exemplary embodiment, the at least five additional transgenes include at least two anticoagulation factors and at least two immunosuppressive factors. In one embodiment, the animal can be modified to express a complement regulatory factor peptide, a biologically active portion or derivative thereof. In one embodiment, the complement regulatory factor peptide is a full-length complement regulatory factor. In a further embodiment, the complement regulatory factor peptide can contain a complement regulatory protein less than full length.

[0272] Any human or porcine complement regulatory factor sequence known to those of skill in the art, or a biologically active portion or fragment thereof, can be used in the compositions and methods of the present invention. In a further embodiment, any consensus complement regulatory factor peptide can be used in accordance with the present invention. In another embodiment, the nucleic acid and / or peptide sequence is at least 80%, 85%, 90%, or 95% homologous to the complement regulatory factor peptides and nucleotide sequences described herein. In a further embodiment, any fragment or homologous sequence that exhibits activity similar to a complement regulatory factor can be used.

[0273] Optionally, an animal that lacks expression of alpha1,3gal and expresses at least one complement regulatory factor (e.g., a complement inhibitory factor) among at least four transgenes has at least one additional genetic modification.

[0274] B. Immunosuppressive factors In one embodiment, the present invention provides a transgenic animal suitable for use as a source of organs, tissues, and cells for xenotransplantation, wherein the donor animal is genetically modified to incorporate and express at least one immunosuppressive factor. The transgenic animal generally has one or more additional genetic modifications, more particularly five or more additional genetic modifications, and even more particularly six or more additional genetic modifications.

[0275] The "immunosuppressive" transgene is capable of downregulating the immune response. For any type of transplantation procedure, the balance between efficacy and toxicity is a key factor for its clinical acceptance. Regarding islet transplantation, a further concern is that many of the current immunosuppressive agents, particularly glucocorticoids or calcineurin inhibitors such as Tacrolimus, damage beta cells or induce peripheral insulin resistance (Zeng et al., Surgery (1993) 113:98 - 102). A steroid - free immunosuppressive protocol ("Edmonton protocol") including sirolimus, low - dose tacrolimus, and monoclonal antibodies (mAb) against the IL - 2 receptor has been used in a single - center trial of islet transplantation for type 1 diabetic patients (Shapiro, A. M. J. et al., (2000), N. Eng. J. Med., 343:230 - 238). The recent success using the "Edmonton protocol" has renewed enthusiasm for the use of islet transplantation to treat diabetes. However, concerns regarding the toxicity of tacrolimus may limit the application of this therapy in humans.

[0276] Biological agents that block key T - cell co - stimulatory signals, particularly the CD28 pathway, are potential alternatives for protecting islets. Examples of agents that block the CD28 pathway include, but are not limited to, soluble CTLA4, including the mutant CTLA4 molecule.

[0277] T cell activation is involved in the etiology of transplant rejection. T cell activation requires at least two sets of signaling events. The first is initiated by the specific recognition of an antigenic peptide through the T cell receptor in combination with a major histocompatibility complex (MHC) molecule on an antigen-presenting cell (APC5). The second set of signals is antigen-nonspecific and is delivered by T cell co-stimulatory receptors that interact with their ligands on the APC. In the absence of co-stimulation, T cell activation is impaired or interrupted, which can lead to antigen-specific unresponsiveness, a state of clonal anergy, or deletion by apoptotic death. Thus, blockade of T cell co-stimulation can provide an approach for antigen-specific suppression of unwanted immune responses while maintaining normal immune function. (Dumont, F. J., 2004, Therapy, 1:289-304).

[0278] Of the several T cell co-stimulation pathways identified to date, the most prominent is the CD28 pathway. The cell surface molecule CD28 expressed on T cells and its counter-receptor B7.1 (CD80) and B7.2 (CD86) molecules present on dendritic cells, macrophages, and B cells are characterized and identified as attractive targets for interfering with T cell co-stimulation signals. A second T cell surface molecule homologous to CD28 is known as cytotoxic T lymphocyte-associated protein (CTLA4). CTLA4 is a cell surface signaling molecule, but in contrast to the action of CD28, CTLA4 negatively regulates T cell function. CTLA4 has a 20-fold higher affinity for the B7 ligand than CD28. The gene for human CTLA4 was cloned in 1988 and chromosomally mapped in 1990 (Dariavach et al., Eur. J. Immunol. 18:1901-1905 (1988) ; Lafage-Pochitaloff et al., Immunogenetics, 31:198-201 (1990); U.S. Patent No. 5,977,318).

[0279] The CD28 / B7 pathway has emerged as an attractive target for interfering with T cell costimulatory signals. The design of CD28 / B7 inhibitors has exploited the endogenous negative regulator of this system, CTLA4. CTLA4-immunoglobulin (CTLA4-Ig) fusion proteins have been widely studied as a means of inhibiting T cell costimulation. An elusive balance must be achieved with any immunosuppressive therapy; it must provide sufficient suppression to overcome the disease or rejection, but excessive immunosuppression will inhibit the overall immune system. The immunosuppressive activity of CTLA4-Ig has been demonstrated in preclinical studies in animal models of organ transplantation and autoimmune diseases. Soluble CTLA4 has recently been tested in human patients with renal insufficiency, psoriasis, and rheumatoid arthritis and formulated as a drug developed by Bristol-Myers Squibb (abatacept, soluble CTLA4-Ig), which was approved for the treatment of rheumatoid arthritis. This drug is the first in a new class of selective T cell costimulation modulators. Bristol-Myers Squibb is also conducting a Phase II clinical trial with belatacept (LEA29Y) for allogeneic kidney transplantation. LEA29Y is a mutant form of CTLA4 that has been engineered to have a higher affinity for the B7 receptor fused to immunoglobulin than wild-type CTLA4. Repligen Corporation is also conducting a clinical trial with its CTLA4-Ig for idiopathic thrombocytopenic purpura. U.S. Patent No. 5,730,403 describes the use of soluble CTLA4-Ig and CTLA4 mutant molecules for protecting allogeneic islet grafts.

[0280] CTLA-4 from one organism can bind to B7 from another organism, but the highest binding affinity is found with allogeneic B7. Thus, soluble CTLA-4 from the donor organism can therefore bind to both recipient B7 (on normal cells) and donor B7 (on xenograft cells), but it preferentially binds to B7 on the xenograft. Thus, in embodiments of the invention that include porcine animals or cells for xenotransplantation, porcine CTLA4 is common. PCT Publication No. WO99 / 57266 by Imperial College describes porcine CTLA4 sequences and administration of soluble CTLA4-Ig for xenotransplantation therapy. Vaughn A. et al., J Immunol (2000) pp. 3175-3181 describe the binding and function of soluble porcine CTLA4-Ig. Porcine CTLA4-Ig binds to porcine (not human) B7, blocks CD28 on recipient T cells, and renders these local T cells anergic without causing broad-spectrum T cell immunosuppression (Mirenda et al., Diabetes, 54:1048-1055, 2005 See).

[0281] Much of the research on CTLA4-Ig as an immunosuppressive agent has focused on administering the soluble form of CTLA4-Ig to patients. Transgenic mice engineered to express CTLA4-Ig were created and subjected to several series of experiments. Ronchese et al. generally examined immune system function after expression of CTLA4 in mice (Ronchese et al., J Exp Med (1994) 179:809; Lane et al., J Exp Med. (1994) March 1 ; 179(3):819). Sutherland et al. (Transplantation. 2000 69(9):1806-12) described the protective effect of CTLA4-Ig secreted by transgenic fetal pancreatic allografts in mice to test the effect of transgenicly expressed CTLA4-Ig on allogeneic islet transplantation. Lui et al. (J Immunol Methods (2003, 277: 171 - 183) reported the production of transgenic mice expressing CTLA4-Ig under the control of a mammary-specific promoter to induce the expression of soluble CTLA4-Ig in the milk of transgenic animals for use as bioreactors.

[0282] PCT Publication No. WO01 / 30966 by Alexion Phamaceuticals Inc. describes chimeric DNA constructs containing the T cell inhibitor CTLA-4 attached to the complement protein CD59, as well as cells, tissues, and organs of transgenic pigs containing the same. PCT Publication No. WO2007035213 (Revivicor) describes transgenic pig animals genetically modified to express CTLA4-Ig.

[0283] Additional immunosuppressive factors can be expressed in animals, tissues, or cells. For example, genes inactivated in mice to produce an immunodeficient phenotype can be cloned and disrupted by gene targeting in pigs. Some of the genes targeted in mice and that can be targeted to produce immunodeficient pigs include beta2-microglobulin (MHC class I deficiency, Koller et al., Science, 24 8: 1227 - 1230), TCR alpha, TCR beta (Mombaerts et al., Nature , 360: 225 - 231), RAG-1 and RAG-2 (Mombaerts et al., (19 92) Cell 68, 869 - 877, Shinkai et al., (1992) Cell 68, 85 5 - 867, U.S. Patent No. 5,859,307).

[0284] In one embodiment, the donor animal is modified to transgenically express cytotoxic T lymphocyte-associated protein 4-immunoglobulin (CTLA4). The animal or cell can be modified to express a CTLA4 peptide or a biologically active fragment thereof (e.g., an extracellular domain, a truncated form of the peptide with at least the transmembrane domain removed) or a derivative. The peptide can be, for example, human or porcine. The CTLA4 peptide can be mutated. The mutated peptide can have a higher affinity for porcine and / or human B7 molecules than the wild type. In one specific embodiment, the mutated CTLA4 can be CTLA4(Glu104, Tyr29). The CTLA4 peptide can be modified to be expressed intracellularly. Other modifications of the CTLA4 peptide include the addition of an endoplasmic reticulum retention signal to the N or C terminus. The endoplasmic reticulum retention signal can be, for example, the sequence KDEL. The CTLA4 peptide can be fused to a peptide dimerization domain or an immunoglobulin (Ig) molecule. The CTLA4 fusion peptide can include a linker sequence capable of linking two peptides. In another embodiment, an animal lacking the expression of functional immunoglobulins produced by the present invention can be administered a CTLA4 peptide or a variant thereof (pCTLA4-Ig or hCTLA4-Ig (abatacept / Orencia or belatacept)) as a drug to suppress their T cell responses. As used herein, CTLA4 is used to refer to any of these variants or those known in the art, such as CTLA4-Ig.

[0285] In one embodiment, the CTLA4 peptide is full-length CTLA4. In a further embodiment, the CTLA4 peptide can contain a CTLA4 protein less than full-length. In one embodiment, the CTLA4 peptide can contain the extracellular domain of the CTLA-4 peptide. In a particular embodiment, the CTLA4 peptide is the extracellular domain of CTLA4. In a further embodiment, the present invention provides a mutant form of CTLA4. In one embodiment, the mutant form of CTLA4 can have a higher affinity for porcine and / or human B7 than the wild type. In one specific embodiment, the mutated CTLA4 can be human CTLA4 (Glu104, Tyr29).

[0286] In one embodiment, CTLA4 can be a truncated form of CTLA4, where at least the transmembrane domain of the protein is removed. In another embodiment, the CTLA4 peptide can be modified to be expressed intracellularly. In one embodiment, a Golgi retention signal can be added to the N or C terminus of the CTLA4 peptide. In one embodiment, the Golgi retention signal can be the sequence KDEL, which can be added to the C or N terminus of the CTLA4 peptide. In a further embodiment, the CTLA4 peptide can be fused to a peptide dimerization domain. In one embodiment, the CTLA4 peptide can be fused to an immunoglobulin (Ig). In another embodiment, the CTLA4 fusion peptide can contain a linker sequence capable of linking two peptides.

[0287] Any human CTLA4 sequence known to those skilled in the art, or biologically active portions or fragments thereof, can be used in the compositions and methods of the present invention. Non-limiting examples include, but are not limited to, the following Genbank accession numbers that describe human CTLA4 sequences: NM005214.2; BC074893.2; BC074842.2; AF414120.1; AF414120; AY402333; AY209009.1; BC070162.1; BC069566.1; L15006.1; AF486806.1; AC010138.6; AJ535718.1; AF225900.1; AF225900; AF411058.1; M37243.1; U90273.1; and / or AF316875.1. Additional nucleotide sequences encoding CTLA4 peptides can be selected from, but are not limited to, the following Genbank accession numbers from EST databases: CD639535.1; A1733018.1; BM997840.1; BG536887.1; BG236211.1; BG058720.1; A1860i99.1; AW207094.1; AA210929.1; A1791416.1; BX113243.1; AW515943.1; BE837454.1; AA210902.1; BF329809.1; A1819438.1; BE837501.1; BE837537.1; and / or AA873138.1.

[0288] In further embodiments, any consensus CTLA4 peptide can be used in accordance with the present invention. In another embodiment, the nucleic acid and / or peptide sequence is at least 80%, 85%, 90% or 95% homologous to the native CTLA4 peptide and nucleotide sequences. In further embodiments, any fragment or homologous sequence that exhibits activity similar to CTLA4 can be used.

[0289] In other embodiments, the amino acid sequence exhibiting T cell inhibitory activity may be amino acids 38 to 162 of the porcine CTLA4 sequence or amino acids 38 to 161 of the human CTLA4 sequence (see, for example, PCT Publication No. WO01 / 30966). In one embodiment, the portion used should have at least about 25%, preferably at least about 50% of the activity of the parental molecule.

[0290] In other embodiments, the CTLA4 nucleic acids and peptides of the invention can be fused to immunoglobulin genes and their molecules or fragments or regions. References to the CTLA4 sequences of the invention include sequences fused to immunoglobulins. In one embodiment, the Ig may be a human Ig. In another embodiment, the Ig may be IgG, particularly, IgG1. In another embodiment, the Ig may be the constant region of IgG. In a particular embodiment, the constant region may be the Cγ1 chain of IgG1. In one particular embodiment of the invention, the extracellular domain of porcine CTLA4 can be fused to human Cγ1 Ig. In another particular embodiment, the extracellular domain of human CTLA4 can be fused to IgG1 or IgG4. In a further particular embodiment, the extracellular domain of mutant CTLA4 (Glu104, Tyr29) can be fused to IgG1.

[0291] In one embodiment, at least one of the transgenes is B7-H4, also known as B7x. B7-4H was identified in 2003 and belongs to the B7 family of immunoglobulins. See Sica, GL, Immunity, Vol. 18, pp. 849 - 861, June 2003.

[0292] In one embodiment, the donor animal is modified to express transgenicly class II transactivator (CIITA) and its mutants PDL1, PDL2, tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL, CD95L) integrin-associated protein (CD47), HLA-E, HLA-DP, HLA-DQ or HLA-DR.

[0293] Class II transactivator (CIITA) is a bifunctional or multifunctional domain protein that acts as a transcriptional activator and plays a crucial role in the expression of MHC class II genes. A mutant form of the human CIITA gene encoding a protein lacking the first 151 amino acids at the amino terminus has been previously demonstrated to act as a potent dominant negative suppressor of HLA class II expression (Yun et al., Int Immunol. October 1997; 9(10): 1545-53). Porcine MHC class II antigens are potent stimulators of direct T cell recognition by human CD4+ T cells and thus may play an important role in the rejection response to transgenic porcine donors in clinical xenotransplantation. One mutant human CIITA construct has been reported to be effective in porcine cells and significantly suppress IFN[gamma] induction as well as constitutive porcine MHC class II expression. Furthermore, a stably transfected porcine vascular endothelial cell line having the mutant human CIITA construct was unable to stimulate direct T cell xenorecognition by purified human CD4+ T cells (Yun et al., Transplantation, March 15, 2000; 69(5): 940-4). Organs, tissues and cells from CIITA-DN transgenic animals were able to induce a greatly reduced T cell rejection response in human recipients. In combination with other transgenes, transgenic expression of mutant CIITA may enable long-term xenograft survival with clinically acceptable levels of immunosuppression.

[0294] In one embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising (i) a lack of expression of alphaGal; and (ii) a genetic modification that results in the integration and expression of at least two transgenes at a single locus, wherein at least four transgenes comprise at least one immunosuppressive factor. The single locus can be selected from a natural locus, a modified natural locus or a transgenic locus. Optionally, the transgenic animal comprises one or more additional genetic modifications.

[0295] In an exemplary embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that includes (i) a lack of expression of alpha Gal; and (ii) a genetic modification that results in the integration and expression of at least four transgenes at a single locus, wherein at least two of the at least two transgenes are immunosuppressive factors. The single locus can be selected from a natural locus, a modified natural locus, or a transgenic locus. The at least four transgenes can be provided as an MCV and integrated into the locus using a gene editing tool. Optionally, the transgenic animal includes one or more additional genetic modifications.

[0296] In an exemplary embodiment, provided are animals (and organs, tissues, and cells derived therefrom) that lack (or have reduced expression of) functional alpha GT alpha Gal and are genetically modified to integrate and express at least four transgenes at a single locus, wherein the at least four transgenes include at least one immunosuppressive factor. The immunosuppressive factor can be, for example, CIITA-DN or CLTA4-IG. The at least four transgenes can include additional transgenes selected from a complement inhibitory factor, an anticoagulant factor, or a combination thereof. The single locus can be selected from a natural locus, a modified natural locus, or a transgenic locus. The at least three transgenes can be provided as an MCV and integrated into the locus using a gene editing tool. Optionally, the transgenic animal includes one or more additional genetic modifications.

[0297] In an exemplary embodiment, the functional alpha GT alpha Gal expression is lacking (or reduced), and the animal (as well as organs, tissues, and cells derived therefrom) is genetically modified to incorporate and express at least four transgenes at a single locus, where the at least four transgenes include at least two immunosuppressive factors. The immunosuppressive factors can be, for example, CIITA-DN or CLTA4-IG. The at least four transgenes can also include a complement inhibitor, an anticoagulant factor, or a combination thereof. The single locus can be selected from a natural locus, a modified natural locus, or a transgenic locus. The at least three transgenes can be provided as an MCV and can be incorporated into the locus using a gene editing tool. Optionally, the transgenic animal includes one or more additional genetic modifications.

[0298] C. Other immunomodulatory factors PDL1 and PDL2: Common costimulatory molecules for T cell activation are CD80 / 86 or CD40. In addition to these positive costimulatory pathways over the past few years, new costimulatory pathways that mediate negative signals and are important for the regulation of T cell activation have been discovered. One of these newer pathways is the pathway consisting of the programmed death 1 (PD-1) receptor and its ligands PD-L1 and PD-L2. The PD-1 receptor is not expressed in resting cells but is upregulated after T and B cell activation. PD-1 contains a tyrosine-based switch motif of cytoplasmic immunoreceptors, and the binding of PD-L1 or PD-L2 to PD-1 leads to inhibitory signals in T cells. Recent data suggest that the PD1 / PD ligand pathway can play a role in the control of T cell subsets showing regulatory activity. In mice, PD-1 signaling has been shown to be required for the suppressive activity of regulatory T cells (Tregs) and the generation of adaptive Tregs. These observations suggest that PD-1 / PDLig and interactions can not only inhibit T cell responses but also cause immunomodulation. A number of lines of evidence demonstrate that the PD-1 / PD ligand pathway can control allograft engraftment and rejection, implying that these molecules are interesting targets for immunomodulation after organ transplantation. Indeed, gene transfer of PDL1 Ig into donor hearts in a rat transplantation model was able to extend allograft survival. Furthermore, it has also been reported that enhancing PD-1 signaling by injection of PD-L1 Ig protects grafts from rejection in mice. Recent data also show that overexpression of PD-L1 IG in islet grafts in mice can partially extend islet graft survival. Transgenic expression of human PD-L1 or PD-L2 in porcine cells and tissues should reduce the initial human anti-porcine T cell response initiated through the direct sensitization pathway (Plege et al., Transplantation, April 15, 2009; 87(7):975-982).It may also be possible to control T cells sensitized to xenografts through an indirect pathway required to achieve long-term sustained tolerance by induction of Tregs.

[0299] In certain embodiments, transgenic animals lacking expression of alphaGal and incorporating and expressing at least four transgenes under the control of at least two promoters include the incorporation and expression of PDL1 or PDL2.

[0300] TRAIL / Fas L: Expression of apoptosis-inducing ligands such as Fas ligand (FasL, CD95L) or tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL, Apo-2L) can eliminate T cells that attack xenografts. TRAIL is a type II membrane protein with an extracellular domain homologous to that of other tumor necrosis factor family members, which shows the highest amino acid identity (28%) to FasL. TRAIL preferentially exerts its apoptosis-inducing effect on tumor cells. In normal cells, binding of TRAIL receptors does not lead to cell death. Recent studies have shown that the cytotoxic effects of immune cells, including T cells, natural killer cells, macrophages, and dendritic cells, are at least partially mediated by TRAIL. Expression of human TRAIL in transgenic pigs can provide a rational strategy for protecting porcine tissues from cell-mediated rejection after xenotransplantation to primates. Stable expression of human TRAIL has been achieved in transgenic pigs, and the expressed TRAIL has been shown to be biologically functional in vitro (Klose et al., Transplantation, July 27, 2005; 80(2):222-30). (d) CD47: CD47, known as integrin-associated protein, is a ubiquitously expressed 50 kDa cell surface glycoprotein that serves as a ligand for the inhibitory immune receptor SIRPα (also known as CD172a, SHPS-1) on macrophages. CD47 and SIRPα constitute a cell-cell communication system (CD47-SIRPα system) that plays important roles in various cell processes, including cell migration, B cell adhesion, and T cell activation. Furthermore, the CD47-SIRPα system is linked to the negative regulation of phagocytosis by macrophages. CD47 on the surface of some cell types (i.e., erythrocytes, platelets, or leukocytes) can protect against phagocytosis by macrophages by binding to the inhibitory macrophage receptor SIRPα.The role of the CD47-SIRPα interaction in self-recognition and the inhibition of phagocytosis was exemplified by the observation that primary wild-type mouse macrophages rapidly phagocytose non-opsonized RBCs obtained from CD47-deficient mice, whereas those from wild-type mice do not. It has also been reported that CD47 inhibits both Fcγ- and complement receptor-mediated phagocytosis through its SIRPα receptor. It has been demonstrated that porcine CD47 does not induce SIRPα tyrosine phosphorylation in a human macrophage-like cell line and that soluble human CD47-Fc fusion protein inhibits the phagocytic activity of human macrophages against porcine cells. It has also been shown that the manipulation of porcine cells for the expression of human CD47 severely reduces the susceptibility of the cells to phagocytosis by human macrophages (Ide et al., Proc Natl Acad Sci USA, March 20, 2007; 104(12):5062-6). Expression of human CD47 on porcine cells can provide inhibitory signaling to SIRPα on human macrophages, providing an approach to prevent macrophage-mediated xenograft rejection.

[0301] In certain embodiments, the transgenic animal that lacks the expression of alpha Gal and incorporates and expresses at least four transgenes under the control of at least two promoters includes the incorporation and expression of TRAIL or Fas L.

[0302] NK cell response. HLA-E / beta2 microglobulin and HLA-DP, HLA-DQ, HLA-DR: Human natural killer (NK) cells are representative of potential obstacles to the success of xenotransplantation from pigs to humans, as they infiltrate pig organs perfused with human blood ex vivo and lyse pig cells directly and by antibody-dependent cell-mediated cytotoxicity both in vitro and in the presence of human serum. NK cell autoreactivity is prevented by the expression of major histocompatibility complex (MHC) class I ligands of inhibitory NK receptors on normal autologous cells. The inhibitory receptor CD94 / NKG2A, expressed on most activated human NK cells, binds specifically to human leukocyte antigen (HLA)-E. The non-classical human MHC molecule HLA-E is a potent inhibitory ligand for NK cells bearing CD94 / NKG2A and, unlike classical MHC molecules, does not induce allogeneic T cell responses. HLA-E assembles in the endoplasmic reticulum and is transported to the cell surface as a stable trimeric complex consisting of the HLA-E heavy chain beta2-microglobulin (beta2m) and peptides derived from the leader sequences of several MHC class I molecules. Expression of HLA-E has been shown to provide partial protection from the cytotoxicity of xenogeneic human NK cells (Weiss et al., Transplantation, January 15, 2009; 87(1):35-43). Transgenic expression of HLA-E on pig organs has the potential to substantially mitigate the human NK cell-mediated rejection of pig xenografts without the risk of allogeneic responses. Furthermore, transgenic pigs bearing other HLA genes have been successfully generated for the purpose of "humanizing" pig organs, tissues, and cells (Huang et al., Proteomics, November 2006; 6(21):581 See also U.S. Patent No. 6,639,122, pages 5-25.)

[0303] In certain embodiments, the transgenic animal lacking expression of alphaGal and incorporating and expressing at least four transgenes under the control of at least two promoters includes the incorporation and expression of HLA-3.

[0304] CD47: CD47 (cluster of differentiation 47), also known as integrin-associated protein (IAP), is a transmembrane protein encoded by the CD47 gene in humans. CD47 is both an immunosuppressive factor and an immunomodulatory factor and is known to be immunogenic for SIRP alpha signaling.

[0305] In an exemplary embodiment, there is provided an animal (and organs, tissues and cells derived therefrom) that lacks (or has reduced expression of) functional alphaGT alphaGal and is genetically modified to incorporate and express at least four transgenes at a single locus, wherein at least one of the at least four transgenes is CD47. The at least four transgenes can include additional transgenes selected from complement inhibitors, anticoagulants, or combinations thereof. The single locus can be selected from a natural locus, a modified natural locus, or a transgenic locus. At least three transgenes can be provided as MCVs and incorporated into the locus using gene editing tools. Optionally, the transgenic animal includes one or more additional genetic modifications.

[0306] In an exemplary embodiment, there is provided an animal (and organs, tissues and cells derived therefrom) that lacks (or has reduced expression of) functional alphaGT alphaGal and is genetically modified to incorporate and express at least four transgenes at a single locus, wherein at least one of the at least four transgenes is CD7. The at least four transgenes can include additional transgenes selected from complement inhibitors, anticoagulants, or combinations thereof. The single locus can be selected from a natural locus, a modified natural locus, or a transgenic locus. At least three transgenes can be provided as MCVs and incorporated into the locus using gene editing tools. Optionally, the transgenic animal includes one or more additional genetic modifications.

[0307] (ii) Anticoagulation factor In one embodiment, the present invention provides a transgenic donor animal suitable for use as a source of organs, tissues, and cells for xenotransplantation, wherein the donor animal is genetically modified to incorporate and express at least one anticoagulation factor. The animal generally has additional genetic modifications, more specifically at least 5 additional genetic modifications, and even more specifically at least 6 additional genetic modifications. In an exemplary embodiment, the present invention includes (i) the absence of expression of alpha Gal; and (ii) genetic modifications that result in the incorporation and expression of at least 4 transgenes at a single locus under the control of at least 2 promoters, wherein at least one transgene is an anticoagulation factor transgenic animal.

[0308] The anticoagulation factor may be any suitable anticoagulation factor. The expression may be ubiquitous or tissue-specific. In certain embodiments, the expression is controlled by a promoter that is primarily active in endothelial cells.

[0309] Representative non-limiting examples of suitable anticoagulation factor transgenes include tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor (EPCR), CD39, and combinations thereof.

[0310] Tissue factor pathway inhibitor (TFPI) is a single-chain polypeptide that can reversibly inhibit factor Xa (Xa) and thrombin (factor IIa), and thus inhibits TF-dependent coagulation. For a review of TFPI, see Crawley and Lane (Arterioscler Thromb Vasc Biol. 2008;28(2):233-42). Dorling and colleagues generated transgenic mice expressing a fusion protein consisting of three Kunitz domains of human TFPI linked to the transmembrane / cytoplasmic domain of human CD4 with a P-selectin tail for targeting to Weibel-Palade intracellular storage granules (Chen D et al., Am J Transplant. 2004;4:1958-1963). The resulting activation-dependent presentation of TFPI on the endothelium was sufficient to completely inhibit thrombus-mediated acute humoral rejection of mouse heart allografts by cyclosporine-treated rats. There was also a suggestion that effective regulation of coagulation could prevent chronic rejection. Similar results were obtained in transgenic mouse hearts expressing hirudin / CD4 / P-selectin fusion protein, indicating that inhibition of thrombin generation or activity is key to protection in this model.

[0311] Hirudin is a naturally occurring peptide in the salivary glands of medicinal leeches (such as Hirudo medicinalis) and is a potent inhibitor of thrombin. Dorling and co-workers Researchers (Chen et al., J Transplant. December 2004; 4(12):1958 - 63) also generated transgenic mice expressing a membrane - linked hirudin fusion protein and transplanted their hearts into rats (mouse - rat xenograft - Tx). In contrast to control non - transgenic mouse hearts, which were all rejected within 3 days, when T - cell - mediated rejection was inhibited by the administration of cyclosporine A, 100% of the organs from both strains of transgenic mice were completely resistant to humoral rejection and survived for over 100 days. Riesbeck et al. (Circulation. December 15, 1998; 98(24):2744 - 52) also investigated the expression of hirudin fusion proteins in mammalian cells as a strategy for the prevention of intravascular thrombosis. Expression in cells reduced local thrombin levels and inhibited fibrin formation. Thus, hirudin is another anticoagulant transgene of interest for preventing the thrombotic effects present in xenotransplantation.

[0312] Thrombomodulin (TM) functions as a cofactor in the thrombin - induced activation of protein C in the anticoagulant pathway by forming a 1:1 stoichiometric complex with thrombin. Endothelial protein C receptor (EPCR) is an N - glycosylated type I membrane protein that enhances the activation of protein C. The roles of these proteins in the protein C anticoagulant system have been reviewed by Van de Wouwer et al. (Arterioscler Thromb Vasc Biol. August 2004; 24(8):1374 - 83). The expression of these and other anticoagulant transgenes has been investigated by various groups to potentially address the coagulation barrier to xenotransplantation (reviewed by Cowan and D'Apice, Cur Opin Organ Transplant, April 2008; 13(2):178 - 83). Esmon and co - workers (Li et al., J Thromb Haemost. July 2005; 3(7):135 Pages 1 to 9) showed that overexpression of EPCR on the endothelium of transgenic mice protects such mice from the challenges of thrombosis. Iino et al. (J Thromb Haemost. May 2004; 2(5): 833 - 4) proposed ex - vivo overexpression of TM in donor islets through gene therapy as a means to prevent thrombotic complications in islet transplantation.

[0313] CD39 is a major vascular nucleoside triphosphate diphosphohydrolase (NTPDase) that converts ATP and ADP to AMP and ultimately to adenosine. Extracellular adenosine plays an important role in thrombosis and inflammation and has thus been studied for its beneficial role in transplantation (reviewed by Robson et al., Semin Thromb Hemost. April 2005; 31(2): 217 - 33). Recent studies have shown that CD39 has a major effect in reducing the inflammatory response (Beldi et al., Front Biosci, 2008, 13: 2588 - 2603). Transgenic mice expressing hCD39 showed impaired platelet aggregation, prolonged bleeding time, and resistance to systemic thromboembolism in a heart transplantation model (Dwyer et al., J Clin Invest., May 2004; 113(10): 1440 - 6). They were also shown to express CD39 on pancreatic islets, and when incubated with human blood, these islets significantly delayed the clotting time compared to wild - type islets (Dwyer et al., Transplantation, August 15, 2006; 82(3): 428 - 32). Preliminary efforts to express hCD39 at high levels from a constitutive promoter system in transgenic pigs showed high post - natal mortality (Revivicor, Inc., unpublished data). However, endothelial cell - specific expression of CD39 has been shown to be better tolerated by transgenic pigs. Therefore, there is a need to express certain anticoagulant transgenes in pigs in a way that does not compromise the well - being of the animals and still provides sufficient levels of expression for clinical xenotransplantation utility.

[0314] In an exemplary embodiment, the invention provides a transgenic animal (e.g., an ungulate, a porcine animal) having a genetic modification that results in (i) absence (or reduced expression) of alpha Gal expression, and (ii) integration and expression of at least four transgenes at a single locus under the control of two promoters, wherein at least one of the at least two transgenes is an anticoagulant factor. In one embodiment, the anticoagulant factor is selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor (EPCR), CD39, and combinations thereof. The single locus may be a natural locus, a modified natural locus, or a transgenic locus. The natural locus may be GGTA1, B4GalNT2, CMAH, Rosa26, AAVS1, or other endogenous locus that can confer beneficial expression characteristics on the integrated transgene. The at least four transgenes under the control of at least two promoters can be provided as an MCV, and the integration can include gene editing tools. Such editing can include targeted insertion into a predetermined site (e.g., a landing pad) that acts as a "safe harbor" (so as not to disrupt any essential genes in the genome) and / or provides desirable features that are specific to the integration site. In the case of insertion at a locus important for preventing xenograft rejection, the insertion of multiple transgenes can have the effect of inactivating porcine genes involved in inducing a xenogeneic response in primates (i.e., inactivation of alpha Gal, CMAH, or B4GalNT2 or others (iGB3, Forssman)). Optionally, the animal can include one or more additional genetic modifications at more than one locus, where at least four transgenes are inserted at one locus and another set of two or more transgenes (under the control of at least two promoters) can be co-integrated at a second site. Alternative embodiments provide MCV insertion at one locus and targeted inactivation at a different locus, and such inactivation can be facilitated by gene editing tools.

[0315] In an exemplary embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) having (i) a lack of (or reduced) expression of alphaGal, and (ii) a genetic modification that results in the integration and expression of at least 4, at least 5, at least 6, at least 7, or at least 8 or more transgenes at a single locus, wherein at least 1, at least 2, or at least 3 of the transgenes are anticoagulant factors.

[0316] In one embodiment, the anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. The at least 4 transgenes can be provided as an MCV, and the integration can include a gene editing tool. The single locus can be a native locus, a modified native locus, or a transgenic locus. Optionally, the animal can include one or more additional genetic modifications.

[0317] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that lacks (or has reduced) expression of alphaGal and is genetically modified to integrate and express at least 3 anticoagulant factors. In certain embodiments, the anticoagulant factors are selected from tissue factor pathway inhibitor (TFPI), hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In certain embodiments, at least 1 of the at least 3 anticoagulant factors is controlled by the expression of a promoter that is predominantly active in endothelial cells. In certain embodiments, at least 2 of the at least 3 anticoagulant factors are controlled by the expression of a promoter that is predominantly active in endothelial cells.

[0318] In an exemplary embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that lacks (or has reduced expression of) the expression of alpha Gal and is genetically modified to incorporate and express at least three anticoagulation factors, wherein at least one of the at least three anticoagulation factors is EPCR.

[0319] In an exemplary embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that lacks (or has reduced expression of) the expression of alpha Gal and is genetically modified to incorporate and express at least three anticoagulation factors, wherein the at least three anticoagulation factors include EPCR and TBM.

[0320] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that lacks (or has reduced expression of) the expression of alpha Gal and is genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes include at least one anticoagulation factor. In certain embodiments, the at least one anticoagulation factor is selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least one anticoagulation factor is EPCR.

[0321] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that lacks (or has reduced expression of) the expression of alpha Gal and is genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes include at least two anticoagulation factors. In certain embodiments, the at least two anticoagulation factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulation factors include EPCR and TBM. In another embodiment, the at least two anticoagulation factors include EPCR and TFPI.

[0322] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that lacks (or has reduced expression of) the expression of alphaGal and is genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes include at least three anticoagulant factors. In certain embodiments, the at least three anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulant factors include EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulant factors include EPCR, TBM, and CD39.

[0323] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that lacks (or has reduced expression of) the expression of alphaGal and is genetically modified to incorporate and express at least five additional transgenes, wherein the at least five additional transgenes include at least two anticoagulant factors. In certain embodiments, the at least two anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulant factors include EPCR and TBM. In another embodiment, the at least two anticoagulant factors include EPCR and TFPI.

[0324] In one embodiment, the present invention provides a transgenic animal (e.g., ungulate, swine) that lacks (or has reduced expression of) alpha Gal and is genetically modified to incorporate and express at least five additional transgenes, wherein the at least five additional transgenes include at least three anticoagulant factors. In certain embodiments, the at least three anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulant factors include EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulant factors include EPCR, TBM, and CD39.

[0325] In one embodiment, the present invention provides a transgenic animal (e.g., ungulate, swine) that lacks (or has reduced expression of) alpha Gal and is genetically modified to incorporate and express at least six additional transgenes, wherein the at least six additional transgenes include at least two anticoagulant factors. In certain embodiments, the at least two anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulant factors include EPCR and TBM. In another embodiment, the at least two anticoagulant factors include EPCR and TFPI. Optionally, the at least six additional transgenes also include at least one immunosuppressive factor.

[0326] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a porcine animal) that lacks (or has reduced expression of) the expression of alphaGal and is genetically modified to incorporate and express at least six additional transgenes, wherein the at least six additional transgenes include at least three anticoagulation factors. In certain embodiments, the at least three anticoagulation factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulation factors include EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulation factors include EPCR, TBM, and CD39.

[0327] (iii) cytoprotective transgene In one embodiment, the present invention provides a transgenic donor animal suitable for use as a source of organs, tissues, and cells for xenotransplantation, wherein the donor animal is genetically modified to incorporate and express at least one cryoprotective factor transgene ("cytoprotective factor"). In an exemplary embodiment, the present invention includes (i) lack of expression of alphaGal; and (ii) genetic modification resulting in the incorporation and expression of at least four transgenes at a single locus under the control of at least two promoters, wherein at least one of the at least four transgenes is a cytoprotective transgene, a transgenic animal (e.g., a pig).

[0328] Cytoprotective transgenes are thought to include anti-apoptosis factors, anti-oxidation factors, and anti-inflammatory factors. Examples include the following: (a) A20: A20 provides anti-inflammatory and anti-apoptotic activities. Vascularized transplanted organs can be protected from endothelial cell activation and cell damage by anti-inflammatory molecules, anticoagulant molecules, and / or anti-apoptotic molecules. Among the genes with great potential for modulation of acute vascular rejection (AVR) is the human A20 gene (hA20), which was first identified as a tumor necrosis factor (TNF)-α-inducible factor in human umbilical vein endothelial cells. Human A20 has a dual cytoprotective function by protecting endothelial cells from TNF-mediated apoptosis and inflammation through blocking several caspases and the transcription factor nuclear factor-κB, respectively. Viable A20 transgenic piglets were produced, and in these animals, the expression of hA20 was limited to skeletal muscle, heart, and PAEC, which were protected from TNF-mediated apoptosis by hA20 expression and were at least partially protected from CD95 (Fas)L-mediated cell death. Furthermore, cardiomyocytes from hA20-transgenic cloned piglets were partially protected from cardiac injury (Oropeza et al. , Xenotransplantation, November 2009; 16(6): 522-34). (b) HO-1: HO provides anti-inflammatory, anti-apoptotic and antioxidant activities. Heme oxygenase (HO), also named HSP32, the rate-limiting enzyme in the heme catabolic action, belongs to the members of heat shock proteins, where the heme ring is cleaved into ferrous iron, carbon monoxide (CO) and biliverdin, which is then converted to bilirubin by biliverdin reductase. Three isoforms of HO, including HO-1, HO-2 and HO-3, have been cloned. The expression of HO-1 is highly inducible, while HO-2 and HO-3 are constitutively expressed (Maines M D et al., Annual Review of Pharmacology & Toxicology, 1997; 37: 517-554 and Choi A M et al., American Journal of Respiratory Cell & Molecular Biology 1996; 15: 9-19). Analysis of HO-1- / - mice suggests that the gene encoding HO-1 regulates iron homeostasis and functions as a cytoprotective gene with potent antioxidant, anti-inflammatory and anti-apoptotic effects (Poss K D et al., Proceedings of the National Academy of Sciences of the United States of America, 1997; 94: 10925-10930, Poss K D et al., Proceedings of the National Academy of Sciences of the United States of America, 1997; 94: 10919-10 924 and Soares M P et al., Nature Medicine, 1998; 4: 1073-1077). Similar findings have recently been described in case reports of HO-1 deficiency in humans (Yachie A et al., Journal of Clinical Investigation, 1999; 103: 1 (pages 29 - 135). The molecular mechanisms responsible for the cytoprotective effects of HO-1, including anti-inflammation, anti-oxidation, and anti-apoptosis, are mediated by its reaction products. HO-1 expression can be modulated in vitro and in vivo by protoporphyrin with different metals. Cobalt protoporphyrin (CoPP) and iron protoporphyrin (FePP) can up-regulate the expression of HO-1. In contrast, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP) inhibit the activity of HO-1 at the protein level. Recently, it has been shown that the expression of HO-1 suppresses the rejection reaction of heart transplantation from mice to rats (Sato K et al., J. Immunol. 2001; 166: 4 (pages 185 - 4194), protects islet cells from apoptosis, and improves the in vivo function of islet cells after transplantation (Pileggi A et al., Diabetes, 2001; 50: 1983 - 1991). Administration of HO-1 by gene transfer provides protection from hyperoxia-induced lung injury (Otterbein L E et al., J Clin Invest, 1999; 103: 1047 - 1054), up-regulation of HO-1 protects genetically fatty Zucker rat livers from ischemia / reperfusion injury (Amersi F et al., J Clin Invest, 1999; 104: 1631 - 1639), and deletion or expression of the HO-1 gene modulates cisplatin-induced renal tubular apoptosis (Shiraishi F et al., Am J Physiol Renal Physiol 2000; 278: F726 - F736) has also been demonstrated. In transgenic animal models, overexpression of HO-1 prevents pulmonary inflammatory and vascular responses to hypoxia (Minamino T et al., Proc. Natl. Acad. Sci. USA , 2001; 98: 8798 - 8803), and protects the heart from ischemia and reperfusion injury (Yet S F et al., Cir Res 2001; 89: 168 - 173) were shown. Pigs having the HO-1 transgene were produced, but no clinical effects related to their use in xenotransplantation were reported (U.S. Patent No. 7,378,569). (c) FAT-1: FAT-1 provides anti-inflammatory activity. Polyunsaturated fatty acids (PUFAs) play a role in inhibiting inflammation (n-3 class). Mammalian cells lack desaturases that convert n-6 to n-3 PUFAs. As a result, essential n-3 fatty acids must be supplied in the diet. However, unlike mammals, the free-living nematode Caenorhabditis elegans expresses an n-3 fatty acid desaturase that introduces a double bond at the n-3 position of the hydrocarbon chain to n-6 fatty acids to form n-3 PUFAs. Transgenic mice were produced that express the C. elegans fat-1 gene and can thus efficiently convert 6-series dietary PUFAs to 3-series PUFAs, such as EPA (20:5 n-3) and DHA (22-6 n-3). (Kang et al., Nature, February 5, 2004; 427(6974):504). Another group generated a transgenic mouse model in which the codons of the fat-1 cDNA were further optimized for efficient translation in the mammalian system; endogenous production of n-3 PUFAs was achieved through overexpression of the C. elegans n-3 fatty acid desaturase gene mfat-1. This group showed that the cellular increase in n-3 PUFAs and reduction in n-6 PUFAs through transgenic expression of mfat-1 enhanced insulin secretion stimulated by glucose, amino acids, and GLP-1 and made the islets highly resistant to cytokine-induced cell death in isolated pancreatic islets of mice (Wei et al., Diabetes, February 2010; 59(2):471-8). (d) Soluble TNF-alpha receptor (sTNFR1): Tumor necrosis factor (TNF, cachexin or cachectin, and formally known as tumor necrosis factor alpha) is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate acute-phase reactions. The main role of TNF is in the regulation of immune cells. TNF can induce apoptotic cell death in order to induce inflammation. Soluble TNF-alpha receptor 1 (sTNFR1) is the extracellular domain of TNFR1 and is an antagonist to TNF-alpha (Su et al., 1998, Arthritis Rheum. 41:139-149). . Transgenic expression of sTNFR1 in xenografts can have beneficial anti-inflammatory effects.

[0329] Other cytoprotective factors with related antioxidant properties include, without limitation, SOD and catalase. Oxygen is an essential molecule for all aerobic organisms and plays a dominant role in ATP production, i.e., oxidative phosphorylation. During this process, reactive oxygen species (ROS), including superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), are produced as by-products. In humans, an antioxidant defense system balances ROS production. Superoxide dismutase (SOD) and catalase are two enzymes with antioxidant properties. SOD catalyzes the disproportionation of superoxide radicals to hydrogen peroxide, which is then converted to water by catalase and glutathione peroxidase. Cellular damage resulting from ROS production can occur in the transplantation setting. Due to reduced antioxidant defense, pancreatic beta cells are particularly vulnerable to free radical and inflammatory damage. Commonly used immunosuppressive drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to the islets and provide little protection from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. Therefore, there is interest in treating islets ex vivo with antioxidant factors or expressing antioxidant genes through gene therapy or transgenic expression in donor tissue. Ex vivo gene transfer of EC-SOD and catalase was anti-inflammatory in a rat model of antigen-induced arthritis (Dai et al., Gene Ther. April 2003;10(7):550-8). Furthermore, delivery of the EC-SOD and / or catalase gene through the portal vein significantly attenuated liver I / R injury in a mouse model (He et al., Liver Transpl. December 2006; 12(12):1869-79). In recent mouse studies, pancreatic islets treated with catalytic antioxidant factors prior to syngeneic, suboptimal syngeneic or xenogeneic transplantation showed superior function compared to untreated controls. In this same study, diabetic mouse recipients of allogeneic islets treated with catalytic antioxidant factors showed improved glycemic control after transplantation and demonstrated a delay in allograft rejection (Sklavos et al., Diabetes, July 2010;59(7):17 Pages 31 - 8, Epub April 22, 2010). In another mouse study, islet grafts overexpressing MnSOD functioned approximately 50% longer than control grafts (Bertera et al., Diabetes, February 2003; 52(2):387 - 93).

[0330] Furthermore, certain anticoagulant factors, including thrombomodulin, EPCR, and CD39, also provide anti - inflammatory activity.

[0331] In an exemplary embodiment, the present invention provides a transgenic animal (e.g., a pig) that includes (i) a lack of expression of alphaGal; and (ii) a genetic modification that results in the integration and expression of at least four transgenes at a single locus (under the control of at least two promoters), where at least one of the at least four transgenes is a cytoprotective transgene. The single locus may be a natural locus, a modified natural locus, or a transgenic locus. The at least two transgenes can be provided as an MCV, and the integration can include gene editing tools. Optionally, the animal can have one or more additional genetic modifications.

[0332] In an exemplary embodiment, the invention provides a transgenic animal (e.g., a pig) that comprises: (i) lack of expression of alphaGal; and (ii) a genetic modification that results in the integration and expression of at least 5, at least 6, at least 7, or at least 8 transgenes at a single locus, or at least 4 transgenes at one locus and one or more transgenes at a second locus, wherein at least one of the transgenes is a cytoprotective transgene, and at least 4 transgenes are under the control of at least 2 promoters, which may be different combinations of constitutive, ubiquitous, tissue-specific, or inducible regulated promoter systems. The transgenes can be provided as MCVs, and the integration can include gene editing tools. The single locus can be a native locus, a modified native locus, or a transgenic locus. Optionally, the animal can have one or more additional genetic modifications.

[0333] D. Production of Transgenic Animals Transgenic animals can be produced by any method known to those of skill in the art, including but not limited to selective breeding, nuclear transfer, introduction of DNA into oocytes, sperm, zygotes, or blastomeres, or use of embryonic stem cells. Gene editing tools can also be utilized, as further described herein.

[0334] In some embodiments, the genetic modification can be identified in animals that are then mated together to form a group of animals having the desired set of genetic modifications (or a single genetic modification). These offspring can be further mated to produce different or the same set of genetic modifications (or a single genetic modification) in their offspring. This cycle of mating to obtain animals having the desired genetic modification(s) can continue as long as desired. In this context, a "group" can include multiple generations of animals produced over time that have the same or different genetic modification(s). A "group" can also refer to a single generation of animals having the same or different genetic modification(s).

[0335] Cells useful for gene modification (e.g., but not limited to, via homologous recombination, random insertion / incorporation, nuclease editing, zinc finger + TALEN nuclease, CRISPR / Cas9 nuclease) include, by way of example, epithelial cells, nerve cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiomyocytes (cardiac muscle cell) and other muscle cells, etc. Further, cells used to produce genetically modified animals (e.g., but not limited to, via nuclear transfer) can be obtained from different organs, such as skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, etc. Cells can be obtained from any cell or organ of the body, including all somatic cells or germ cells.

[0336] Furthermore, animal cells that can be genetically modified can be obtained from various different organs and tissues, such as, for example, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and whole or partial disaggregated preparations of embryos, fetuses or adult animals, but not limited to these. In one embodiment of the present invention, the cells are epithelial cells, fibroblasts, nerve cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiomyocytes (cardiac muscle cell), other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, pancreatic islet cells, blood cells, blood progenitor cells, bone cells (bone cell), bone progenitor cells, neural stem cells, primordial stem cells, adult stem cells, mesenchymal stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, hepatic stellate cells, aortic smooth muscle cells, cardiomyocytes (cardiac myocyte), neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes , pancreatic cells, thyroid cells, parathyroid cells, parotid gland cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen-presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, Leydig cells, peritubular cells, Sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicular cells, mucosal cells, ciliated cells, non-keratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, and osteoclasts, but are not limited to these It can be selected from the group consisting of, but not limited to, these. In an alternative embodiment, embryonic stem cells can be used. An embryonic stem cell line can be used, or embryonic stem cells can be newly obtained from a host such as a porcine animal. These cells can be grown on an appropriate fibroblast-feeder layer or can be grown in the presence of leukemia inhibitory factor (LIF).

[0337] Embryonic stem cells are a preferred germ cell type. An embryonic stem cell line can be used, or embryonic stem cells can be newly obtained from a host such as a porcine animal. These cells can be grown on an appropriate fibroblast-feeder layer or can be grown in the presence of leukemia inhibitory factor (LIF).

[0338] Particularly for cells of interest, among other lineages, stem cells such as hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, etc., pancreatic islets of Langerhans, adrenal medullary cells capable of secreting dopamine, osteoblasts, osteoclasts, epithelial cells, endothelial cells, leukocytes, e.g., B and T lymphocytes, bone marrow monocytic cells, etc., neurons, glial cells, ganglion cells, retinal cells, liver cells, e.g., hepatocytes, bone marrow cells, keratinocytes, hair follicle cells, and myoblast (muscle) cells are included. ​In certain embodiments, these cells can be fibroblasts or fibroblast-like cells that are morphologically or phenotypically indistinguishable from fibroblasts, or have a pre-senescent lifespan of at least 10 or at least 12 or at least 14 or at least 18 or at least 20 days, or a lifespan sufficient to permit homologous recombination and nuclear transfer of non-senescent nuclei; in a specific embodiment, these cells can be fetal fibroblasts. Fibroblasts are a suitable somatic cell type because they can be obtained in large quantities from developing fetuses and adult animals. These cells can be readily expanded in vitro with a rapid doubling time and can be clonally expanded for use in gene targeting procedures. The cells used can be derived from fetal animals, or can originally be neonates, or can be derived from adult animals. These cells can be mature or immature, and can be either differentiated or undifferentiated.

[0339] (i) homologous recombination Homologous recombination allows for site-specific modification in endogenous genes, and thus new changes can be engineered into the genome. The main step in homologous recombination is DNA strand exchange, which involves the pairing of a DNA double strand with at least one DNA strand containing complementary sequences that form an intermediate recombination structure containing heteroduplex DNA (see, for example, Radding, C. M. (1982) Ann. Rev. Genet. 16:405; U.S. Patent No. 4,888,274). This heteroduplex DNA can take several forms including a three-stranded DNA containing a triplex form in which a single complementary strand invades the DNA double strand (Hsieh et al. (1990 (Rao et al., Genes and Development 4:1951 (1990); Rao et al., PNAS 88:2984 (1991)), when two complementary DNA strands pair with a DNA duplex, a classical Holliday recombination junction or chi structure (Holliday, R., Genet. Res. 5:282 (1964)), or a double-D loop (U.S. Patent Application No. 07 / 755,462, filed Sep. 4, 1991, “Diagnostic Applications of Double-D Loop Formation”) can form. Once formed, the heteroduplex structure can be resolved by strand cleavage and exchange such that all or part of the invading DNA strand is spliced into the recipient DNA duplex, adding or replacing segments of the recipient DNA duplex. Alternatively, the heteroduplex structure can give rise to gene conversion, where the sequence of the invading strand is transferred to the recipient DNA duplex by repair of mismatched bases using the invading strand as a template (Genes, 3rd ed. (1987) Lewin, B., John Wiley, New York, N.Y.; Lopez et al., Nucleic Acids Res. 15:5643 (1987)). Whether by the mechanism of cleavage and religation or by the mechanism(s) of gene conversion, formation of heteroduplex DNA at a homologously paired junction can function to transfer genetic sequence information from one DNA molecule to another.

[0340] The ability to transfer genetic sequence information between DNA molecules by homologous recombination (gene conversion and classical strand breakage / religation) makes targeted homologous recombination a powerful method in genetic engineering and gene manipulation.

[0341] In homologous recombination, incoming DNA interacts with a site in the genome containing a substantially homologous DNA sequence and is incorporated there. In non-homologous (“random” or “illicit”) incorporation, incoming DNA is not found at a homologous sequence in the genome, but It is incorporated at another location, one of a number of potential locations. In general, studies using higher eukaryotic cells have revealed that the frequency of homologous recombination is much lower than the frequency of random incorporation. The ratio of these frequencies has a direct impact on "gene targeting" that depends on incorporation via homologous recombination (i.e., recombination between exogenous "targeting DNA" and corresponding "target DNA" in the genome). The present invention can use homologous recombination in cells such as the above cells to inactivate genes or to insert and upregulate or activate genes. The DNA can include at least a portion of the gene(s) at a specific locus, along with the introduction of alterations into at least one copy, optionally both copies, of the native gene(s) to prevent the expression of a functional gene product. This alteration can be an insertion, deletion, exchange, mutation, or a combination thereof. If an alteration is introduced only into one copy of the inactivated gene, cells having a single non-mutated copy of the target gene are amplified and can be subjected to a second targeting step, where the alteration can be the same as or different from the first alteration, usually different, and can overlap with at least a portion of the originally introduced alteration if a deletion or exchange is involved. In this second targeting step, a targeting vector containing different mammalian selectable markers with the same arms of homology can be used. The resulting transformants are screened for the absence of a functional target antigen, and the cell DNA can be further screened to ensure the absence of the wild-type target gene. Alternatively, homozygosity for a particular phenotype can be achieved by mating a heterozygous host for the mutation.

[0342] Several papers have described the use of homologous recombination in mammalian cells. Examples of these papers are Kucherlapati et al. (1984) Proc. Natl. Acad. Sci. USA 81:3153-3157; Kucherlapati et al. (1985) Mol. Cell. Bio. 5:7 Pages 14 - 720; Smithies et al. (1985) Nature 317:230 - 234; Wake et al. (1985) Mol. Cell. Bio. 8:2080 - 2089; Ayares et al. (1985) Genetics 111:375 - 388; Ayares et al. (1986) Mol. Cell. Bio. 7:1656 - 1662; Song et al. (1987) Proc. Natl. Acad. Sci. USA 84:6820 - 6824; Thomas et al. (1986) Cell 44:419 - 428; Thomas and Capecchi, (1987) Cell 51:503 - 512; Nandi et al. (1988) Proc. Natl. Acad. Sci. USA 85:3845 - 3849 ; as well as Mansour et al. (1988) Nature 336:348 - 352; Evans and Kaufman, (1981) Nature 294:146 - 154; Doetschman et al. (198 7) Nature 330:576 - 578; Thoma and Capecchi, (1987) Cell 51:503 - 512; Thompson et al. (1989) Cell 56:316 - 32 1.

[0343] In one embodiment, at least four transgenes incorporated and expressed in the transgenic animal of the present invention are introduced by homologous recombination. In another embodiment, at least one of the four transgenes incorporated and expressed in the transgenic animal of the present invention is introduced by homologous recombination.

[0344] (ii) Random insertion In one embodiment, the DNA encoding the transgene sequence can be randomly inserted into the chromosome of the cell. This random incorporation can result from any method of introducing DNA into the cell known to those skilled in the art. This includes, but is not limited to, electroporation, sonoporation, use of a gene gun, lipotransfection, calcium phosphate transfection, use of dendrimers, microinjection, use of viral vectors including adenovirus, AAV and retroviral vectors, and group II ribozymes. In one embodiment, the DNA encoding can be designed to include a reporter gene such that the presence of the transgene or its expression product can be detected via activation of the reporter gene. Any reporter gene known in the art, such as those disclosed above, can be used. This reporter gene can also be one of the transgenes added to the cell, such that the cell surface expression of that transgene (e.g., DAF or CD46 or EPCR or CD47) can be used in conjunction with flow cytometry (and a fluorescent antibody specific to the transgene) as a means to enrich for gene transfer and subsequence expression of the transgene (and co-inserted transgene combinations). Cells containing the transgene can be selected by selecting cells in which the reporter gene has been activated in cell culture. In other embodiments, the DNA encoding the transgene can be introduced into the cell via electroporation. In other embodiments, the DNA can be introduced into the cell via lipofection, infection or transformation. In one embodiment, electroporation and / or lipofection can be used to transfect fibroblasts. In certain embodiments, the transfected fibroblasts can be used as nuclear donors for nuclear transfer to generate transgenic animals, as known in the art and described below.

[0345] Subsequently, the cells stained for the presence of the reporter gene can be sorted by FACS to enrich the cell population such that the inventors have a higher percentage of cells containing the DNA encoding the gene of interest. In other embodiments, the FACS-sorted cells can then be cultured for a period of time such as 12, 24, 36, 48, 72, 96 hours or longer, or for a period of time that allows the DNA to be incorporated, to obtain a population of stably transfected cells.

[0346] In one embodiment, at least four transgenes incorporated and expressed in the transgenic animals of the present invention are introduced by random integration. In another embodiment, at least one of the four transgenes incorporated and expressed in the transgenic animals of the present invention is introduced by random integration. For example, a dicistronic vector containing at least two transgenes is integrated into the genome by random integration.

[0347] (iii) Targeted genome editing: In an exemplary embodiment, the transgene is incorporated into the animal using a genome editing tool. These tools include, but are not limited to, nucleases and site-specific recombinases. In an exemplary embodiment, the method of insertion is facilitated by a genome editing method that utilizes a gene editing tool such as, but not limited to, integrase (recombinase), CRISPR / CAS9 nuclease, TALAN nuclease, zinc finger nuclease.

[0348] The transgene can be targeted to a single locus selected from a native locus, a modified native locus, or a transgenic locus (e.g., a landing pad). Native loci can be, for example, GGTA1, β4GalNT2, CMAH, ROSA26, AAVS1. The native locus can be modified, i.e., a modified native locus, for example, a modified (GGTA1, β4GalNT2 or CMAH).

[0349] In an exemplary embodiment, the transgene can be targeted to a landing pad and / or a docking site or other stable expression site. In one embodiment, a landing pad or docking vector can be inserted into any locus of interest, such as GGTA1, CMAH, β4Gal, ROSA26, AAVS1, or the transgene can be targeted to any known "safe harbor" locus, or any predetermined locus that can provide an advantageous gene expression profile, or the predetermined locus may inactivate a preferred gene where simultaneous insertion and knockout are beneficial for engraftment outcome. In another embodiment, gene editing can be utilized to create a double-strand break, which initiates a DNA repair mechanism to create small insertions, deletions, or nucleic acid substitutions (INDELs) that result in gene activation or knockout at the target site; in such cases, INDELs at one predetermined locus (e.g., GGTA1, CMAH, B4GalNT2) can be created simultaneously in a cell or the resulting cloned pig with a gene editing-enhanced knock-in of a polycistronic vector at another locus.

[0350] In certain embodiments, gene editing is used simultaneously (using one or more Crispr-Cas9 guide RNAs, TALENs, or ZFNs (or combinations thereof)) to inactivate one, two, or three endogenous loci (e.g., one or all of GGTA1, CMAH, B4GalNT2) in the porcine genome, and one or more of these gene editing-enhanced modifications also result in targeted insertion of a polycistronic vector having at least four transgenes under the control of at least two promoters at one or more of such native loci or modified native loci.

[0351] A. Zinc Finger Nucleases / TALENs In one embodiment, the transgene is incorporated using zinc finger nucleases (ZFNs).

[0352] Zinc finger nucleases are a fusion of a non-specific DNA cleavage motif and a sequence-specific zinc finger protein. The nuclease activity is a derivative of the FokI bacterial restriction endonuclease capable of creating single-strand breaks. ZFNs operate by dimerizing two DNA-binding domains together with two FokI enzymes to produce double-strand breaks with 18bp specificity.

[0353] In another embodiment, the transgene is integrated using a transcription activator-like effector nuclease (TALEN).

[0354] TALENs function similarly to ZFNs to create double-strand breaks by tethering the FokI endonuclease to a DNA-binding domain. In this process, the targeting efficiency of mutagenesis directed by TALENs has been reported to reach an efficiency of 73.1% with a biallelic knockout at a rate of 27.8%. TALENs can be distinguished from ZFNs by the ease of gene design, reduced cost, and slightly improved targeting frequency.

[0355] In one embodiment, the present invention utilizes direct injection of ZFNs and TALENs into porcine zygotes that can introduce endogenous genes, or small insertions or deletions or nucleotide substitutions, to produce piglets with desired genetic modifications.

[0356] B. CRISPR / CAS9 nuclease In another embodiment, the transgene is integrated using CRISPR / CAS9 nuclease.

[0357] CRISPR / Cas9 is derived from a bacterial defense mechanism that cleaves exogenous DNA by RNA-guided targeting. In bacteria, foreign DNA is digested and CRISPR The RNA (crRNA) is inserted into the CRISPR locus from which it is made. These short RNA sequences then associate with homologous - presumably foreign - sequences in the genome. If, following the homologous genomic sequence, an appropriate "protospacer adjacent motif" (PAM) follows at the 3' end, the Cas9 endonuclease creates a double - strand break. The PAM spacer helps prevent the CRISPR locus itself from being targeted. The CRISPR / Cas9 system has proven useful outside of bacteria and was first used in 2013 to remove alphaGal from the pig genome. The most commonly used system is derived from Streptococcus pyogenes with a 3' PAM sequence of NGG, where N represents any nucleotide. This system enables the creation of mutagenic events in any porcine genomic sequence consisting of GN 19 NGG.

[0358] The CRISPR / Cas9 system can also be used in conjunction with homology directed repair (HDR), a naturally occurring nucleic acid repair system initiated by the presence of double - strand breaks (DSBs) in DNA (Liang et al. 1998). More specifically, the CRI SPR / Cas9 system can be used to create targeted double - strand breaks and can be used to control the specificity of HDR genome engineering techniques (Findlay et al. 2014 ; Mali et al. February 2014; Ran et al. 2013) and is useful for modifying the genome in many organisms, including mammals and humans (Sander and Joung, 2014).

[0359] After RNA-guided cleavage of a specific site in DNA to create a double-strand break, a DNA fragment or DNA construct of interest can be inserted. This donor template, fragment or construct has the desired insertion or modification with segments of DNA homologous to the blunt ends of the cleaved DNA adjacent. Thus, the cell's native DNA repair machinery can utilize homology-directed recombination, which is known to edit the genome of target cells with high precision and create highly targeted double-strand breaks, in combination with any genome editing technology known to create the desired gene material. Genomic modifications performed in this way can be used to insert a new gene, as described as "enhanced homology-directed insertion or knock-in" as an insertion of DNA, and simultaneously knock out an existing gene (Mali et al., February 2013).

[0360] The CRISPR / Cas system offers several advantages over previous site-specific nucleases. First, the Cas9 endonuclease represents a method of DNA cleavage without an initial tethering. This freely associates with multiple guide RNAs, thereby enabling the simultaneous targeting of several loci within a single transfection. This has enabled the efficient combination of multiple gene knockouts on a single cell. In 2013, the creation of GGTA1, GGTA1 / iGb3S, GGTA1 / CMAH and GGTA1 / iGb3S / CMAH homozygous knockout cells was achieved in a single reaction. The CRISPR / Cas9 system has been successfully used to generate transgenic animals in various vertebrates including zebrafish, monkeys, mice, rats and pigs. See Withworth et al., Biol. Reprod. 91(3):78, 1-13

[2014] and and Li et al., Xenotransplantation 22(1), 20-31

[2015] . for this matter.

[0361] Targeting efficiency, or the percentage of desired mutations achieved, is one of the most important parameters for evaluating genome editing tools. The targeting efficiency of Cas9 is comparable to more established methods such as TALEN or ZFN. For example, in human cells, custom-designed ZFNs and TALENs can only achieve efficiencies in the range of 1% to 50%. In contrast, the Cas9 system has been reported to have efficiencies of up to >70% in zebrafish and plants, and efficiencies in the range of 2 - 5% in induced pluripotent stem cells.

[0362] In one embodiment, the present invention can utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, swine animals) via microinjection of CRISPR specifically designed to target a gene of interest in "in vitro" induced zygotes.

[0363] In another embodiment, the present invention can utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, swine animals) by modifying somatic donor cells with CRISPR specifically designed to target a gene of interest and subsequent somatic cell nuclear transfer (SCNT).

[0364] In another embodiment, the present invention can utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, swine animals) by targeting specific regions / sequences of existing genetic modifications. In a more specific embodiment, the sequence of the neomycin gene sequence is targeted.

[0365] In another embodiment, the present invention can utilize a genome editing system, such as a TALEN, zinc finger or CRISPR / Cas9 system, to generate transgenic pigs (e.g., ungulates, swine animals) by targeting specific regions / sequences of existing genetic modifications. In a more specific embodiment, a single locus that can be a natural locus, a modified natural locus or a transgenic locus (e.g., a landing pad) is targeted.

[0366] In another embodiment, the CRISPR / Cas9 system can be used to generate transgenic pigs (e.g., ungulates, swine animals) by targeting specific regions / sequences of existing genetic modifications through the insertion of a large DNA fragment or construct flanked by DNA arms or segments homologous to the double-strand break using homology-directed recombination.

[0367] C. Site-Specific Recombinases In an exemplary embodiment, the transgene is integrated using a site-specific recombinase. Site-specific recombinase technology is widely used to effect deletions, insertions, translocations and inversions at specific sites in the DNA of cells. This enables DNA modification to be targeted to specific cell types or to be induced by specific external stimuli. This is carried out in both eukaryotic and prokaryotic systems. There are several recombination systems that work efficiently for genetic engineering strategies. The Flp-FRT and Cre-loxP recombinase systems are reversible and thus facilitate both site-specific integration and excision. Integrases that catalyze highly site-specific recombination reactions that result in precise integration, excision and / or inversion of DNA mediate the genome integration process. Serine (ФC31, Bxb1, R4) and tyrosine integrases (λ, P22, HP1) are two major families of integrases currently applied to genome engineering. In a broad sense, the process of site-specific recombination involves the binding of the recombinase(s) to the recombinase substrate(s) to bring them into close proximity via protein-protein interactions. During this process, the substrate is cleaved and the DNA ends are reorganized in a strand exchange reaction, resulting in religation of the DNA backbone to yield the recombinant product. In most cases, serine integrases catalyze highly efficient irreversible recombination using simple att sites.

[0368] To use highly efficient site-specific recombinases, the docking site or landing pad includes an attachment site for the recombinase substrate binding site, such as the att site; or recombination systems such as Flp-FRT and Cre-loxP can be introduced at the desired locus in the cell line and / or animal line. This insertion of the docking vector into the target genome is either via random recombination or homologous recombination. This allows for successive rounds of plasmid incorporation, where the plasmid or vector can contain different transgenes and / or additional DNA sequences. In turn, recombination systems such as Flp / FRT can be used to remove unwanted vector and marker sequences.

[0369] (iv) Vectors for producing transgenic animals Nucleic acid targeting vector constructs can be designed to achieve homologous recombination in cells. In one embodiment, the targeting vector is designed using a promoter trap, where integration at the targeted locus enables the inserted open reading frame of the transgene to utilize an endogenous or native promoter to drive the expression of the inserted gene (or inserted selectable marker; e.g., Neo or Puro). In certain embodiments, the targeting vector is designed using a "poly(A) trap". Unlike promoter traps, poly(A) trap vectors capture a broader spectrum of genes, including those that are not expressed in target cells (i.e., fibroblasts or ES cells). The polyA trap vector contains a constitutive promoter that drives the expression of a selectable marker gene lacking a polyA signal. Exchanging the polyA signal is a splice donor site designed to splice into a downstream exon. In this strategy, the mRNA of the selectable marker gene can be stabilized upon trapping of the polyA signal of the endogenous gene, regardless of its expression state in the target cell. In one embodiment, a targeting vector is constructed that contains a selectable marker lacking a signal for polyadenylation.

[0370] These targeting vectors can be introduced into mammalian cells by any suitable method including, but not limited to, transfection, transformation, virus-mediated transduction or infection with a viral vector. In one embodiment, the targeting vector may include 3' and 5' recombination arms (i.e., flanking sequences) that are homologous to the genomic sequence of interest. The 3' and 5' recombination arms can be designed to flank the 3' and 5' ends of at least one functional region of the genomic sequence. Targeting of the functional region can inactivate it, resulting in the inability of the cell to produce a functional protein. In another embodiment, the homologous DNA sequence may include one or more intron and / or exon sequences. In addition to the nucleic acid sequence, the expression vector may include a selectable marker sequence, such as a hypersensitive green fluorescent protein (eGFP) gene sequence, an initiation and / or enhancer sequence, a poly A-tail sequence, and / or a nucleic acid sequence that provides for expression of the construct in a prokaryotic host cell and / or a eukaryotic host cell. The selectable marker can be located between the 5' recombination arm sequence and the 3' recombination arm sequence.

[0371] Modification of a targeted locus in a cell can be produced by introducing DNA into the cell, where the DNA has homology to the target locus and contains a marker gene that allows for selection of cells containing the incorporated construct. The homologous DNA in the target vector recombines with chromosomal DNA at the target locus. Homologous DNA sequences, 3'recombination arms and 5'recombination arms may flank both sides of the marker gene. Methods for the construction of targeting vectors have been described in the art. See, for example, Dai et al., Nature Biotechnology 20:251-255, 2002; WO00 / 51424. In such examples, the selectable marker gene can be a promoterless neomycin phosphotransferase (Neo) gene that does not result in targeted insertion and expression of Neo (by trapping and utilizing the endogenous porcine alphaGal gene promoter) and also does not result in functional inactivation of the target locus (e.g., GGTA1) from targeted insertion and disruption of the GGTA1 catalytic domain.

[0372] Various enzymes can catalyze the insertion of foreign DNA into the host genome. Viral integrases, transposases, and site-specific recombinases mediate the incorporation of viral genomes, transposons, or bacteriophages into the host genome. The extensive collection of enzymes with these properties can be derived from a wide variety of sources. Retroviruses combine several useful features, including the relative simplicity of their genomes, ease of use, and the ability to be incorporated into the host cell genome, to enable long-term transgene expression in transduced cells or their progeny. Thus, they have been used in numerous gene therapy protocols. Vectors based on lentiviral vectors are attractive candidates for both gene therapy applications and transgenic applications, similar to adeno-associated virus, a small DNA virus (parvovirus) that replicates simultaneously in mammalian cells together with helper viruses such as adenovirus, herpes simplex virus, or human cytomegalovirus. The viral genome consists essentially of only two ORFs (rep, non-structural proteins, and cap, structural proteins), from which (at least) seven different polypeptides are induced by alternative splicing and alternative promoter usage. In the presence of a helper virus, the rep proteins mediate the replication of the AAV genome. Integration, and thus latent viral infection, occurs in the absence of a helper virus. Transposons are also targeted. These are segments of mobile DNA that can be found in various organisms. Active transposons are found in many prokaryotic systems and insects, but functional natural transposons do not exist in vertebrates. The Drosophila P element transposon has been used for many years as a tool for genetic engineering. The Sleeping Beauty transposon was established from non-functional transposon copies found in salmonid fish and is significantly more active in mammalian cells than prokaryotic or insect transposons. Site-specific recombinases are enzymes that catalyze DNA strand exchange between DNA segments that have only a limited degree of sequence homology.These bind to recognition sequences between 30 and 200 nucleotides in length, cleave the DNA backbone, exchange the two DNA duplexes involved, and religate the DNA. In some site-specific recombination systems, a single polypeptide is sufficient to carry out all of these reactions, while other recombinases require a variable number of accessory proteins to perform these tasks. Site-specific recombinases can be clustered into two protein families with distinct biochemical properties, namely tyrosine recombinases (where the DNA is covalently bound to tyrosine residues) and serine recombinases (where the covalent bond occurs at serine residues). The most commonly used enzymes for genome modification approaches are Cre (a tyrosine recombinase from the E. coli bacteriophage P1) and phiC31 integrase (a serine recombinase from the Streptomyces phage phiC31). Several other site-specific recombinases from bacteriophages (including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase and phage TP901-1 integrase, as well as bxb1 integrase) have been successfully used to mediate stable gene insertion into the mammalian genome. Recently, site-specific recombinases have been purified from Streptomyces bacteriophages. The phiC31 recombinase is a member of the resolvase family and mediates phage integration. In this process, the bacteriophage attP site recombines with the corresponding attB site in the bacterial genome. The crossover generates two sites, attL and attR, which are no longer targets for recombinase action in the absence of accessory proteins. This reaction also occurs in mammalian cells and can therefore be used to mediate site-specific integration of therapeutic genes. The site specificity of tyrosine recombinases has been difficult to modify by direct protein engineering manipulations because the catalytic and DNA recognition domains are closely intertwined. Thus, changes in specificity often accompany loss of activity.Serine recombinases can be engineered to be compliant, and hyperactive derivatives of Tn3 resolvase have been modified by replacing the native DBD with the zinc finger domain of the human zinc finger transcription factor Zif268. The DNA site specificity of the resulting chimeric protein, called Z-resolvase, has switched to that of Zif268. Zinc finger proteins can be modified by in vitro protein evolution to recognize any DNA sequence, and thus this approach can enable the development of chimeric recombinases that can incorporate therapeutic genes at precise genomic locations. Methods for enhancing or mediating recombination include combinations of site-specific and homologous recombination, AAV-vector-mediated recombination, and zinc finger nuclease-mediated recombination (see Geurts et al., Science 325:433, 2009).

[0373] As used herein, the term "vector" refers to a nucleic acid molecule (preferably DNA) that provides useful biological or biochemical properties to the inserted nucleic acid. The "expression vectors" according to the present invention include vectors capable of enhancing the expression of one or more molecules inserted or cloned into the vector upon transformation of the vector into a cell. Examples of such expression vectors include phages, autonomously replicating sequences (ARSs), centromeres, and other sequences that can replicate or be replicated in vitro or in cells, or that can carry a desired nucleic acid segment to a desired location within an animal cell. Expression vectors useful in the present invention include chromosome-derived, episome-derived, and virus-derived vectors, such as vectors derived from bacterial plasmids or bacteriophages, and vectors derived from combinations thereof, such as cosmids and phagemids, or virus-based vectors, such as adenoviruses, AAVs, lentiviruses. The vector may have one or more restriction endonuclease recognition sites at which the sequence can be cleaved in a determinable manner without loss of the essential biological function of the vector, and nucleic acid fragments can be spliced to effect its replication and cloning. The vector can further provide, for example, primer sites for PCR, transcription and / or translation initiation and / or regulatory sites, recombination signals, replicons, selectable markers, etc. Clearly, methods for inserting a desired nucleic acid fragment that do not require homologous recombination, translocation, or the use of restriction enzymes (e.g., UDG cloning of PCR fragments (U.S. Patent No. 5,334,575), TA Cloning.RT-PCR, cloning (Invitrogen Corp., Carlsbad, Calif.), but not limited thereto) can also be applied to clone nucleic acids into the vectors used according to the present invention.

[0374] Homozygous cells at the targeted locus can be produced by introducing DNA into the cells, where the DNA has homology to the target locus and contains a marker gene that allows selection of cells containing the integrated construct. The homologous DNA in the target vector recombines with the chromosomal DNA at the target locus. Homologous DNA sequences, 3' recombination arms, and 5' recombination arms may flank both sides of the marker gene. Methods for construction of targeting vectors have been described in the art. See, for example, Dai et al. (2002) Nature Biotechnology 20:251-255; WO00 / 51424, FIG. 6; and Gene Targeting: A Practical Approach. Joyner, A. Oxford University Press, USA; 2nd Edition, February 15, 2000.

[0375] Various constructs can be prepared for homologous recombination at the target locus. Typically, the construct may contain a sequence of at least 25 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp or 50 kbp that is homologous to the target locus.

[0376] Various considerations, such as the size of the target locus, the availability of the sequence, the relative efficiency of double crossover events at the target locus, and the similarity between the target sequence and other sequences, can be involved in determining the degree of homology of the target DNA sequence. The targeting DNA can include DNA that is substantially homologous to the corresponding target sequence in the genome to be modified and that contains sequences adjacent to the desired sequence modification. The substantially homologous sequence can be at least about 95%, 97 - 98%, 99.0 - 99.5%, 99.6 - 99.9% or 100% identical to the corresponding target sequence (excluding the desired sequence modification). The targeting DNA and the target DNA can preferably share a stretch of DNA of at least about 75, 150 or 500 base pairs that is 100% identical. Thus, the targeting DNA can be derived from cells that are closely related to the cell line being targeted; or the targeting DNA can be derived from cells of the same cell line or animal as the cell being targeted.

[0377] Suitable selectable marker genes include, but are not limited to, genes that confer the ability to grow on a specific culture substrate, such as the tk gene (thymidine kinase), or the hprt gene (hypoxanthine phosphoribosyl transferase) that confers the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine); the bacterial gpt gene (guanine / xanthine phosphoribosyl transferase) that allows growth on MAX medium (mycophenolic acid, adenine and xanthine). See Song et al. (1987) Proc. Nat'l Acad. Sci. U.S.A. Vol. 84: pp. 6820 - 6824. See also Sambrook et al. (1989) Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Chapter 16. . Other examples of selectable markers include genes that confer resistance to compounds such as antibiotics, genes that confer the ability to grow on a selected substrate, proteins that produce a detectable signal such as luminescence, for example, genes encoding green fluorescent protein, enhanced green fluorescent protein (eGFP). For example, antibiotic resistance genes, such as the neomycin resistance gene (neo) (Southern, P. and P. Berg, (1982) J. Mol. Appl. Genet. 1:327-341); and a wide variety of such markers including the hygromycin resistance gene (hyg) (Nucleic Acids Research 11:6895-6911 (1983) and Te Riele et al. (1990) Nature 348:649-651) are known and available. Further reporter genes useful in the methods of the present invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta-galactosidase (LacZ), beta-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. A plurality of selectable markers that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, blasticidin, zeocin, methotrexate, phosphinothricin, puromycin and tetracycline are available. Methods for determining the repression of reporter genes are well known in the art and include, but are not limited to, fluorescence quantification methods (e.g., fluorescence spectroscopy, fluorescence-activated cell sorting (FACS), fluorescence microscopy), antibiotic resistance determination.

[0378] Combinations of selectable markers can also be used. To use a combination of markers, the HSV-tk gene can be cloned such that it is outside of the targeting DNA (another selectable marker can be placed on the opposite flank, if desired). After introducing the DNA construct into the cells to be targeted, those cells can be selected on the appropriate antibiotic. Selectable markers can also be used for negative selection. Negative selection markers generally kill the cells in which they are expressed, either because the expression is toxic per se or because they produce a catalyst that results in a toxic metabolite such as herpes simplex virus type I thymidine kinase (HSV-tk) or diphtheria toxin A. Generally, negative selection markers are incorporated into the targeting vector such that they are lost after an accurate recombination event. Similarly, conventional selectable markers such as GFP can be used for negative selection using, for example, FACS sorting, and if expressed at a significant level on the cell surface, the insertion of the selected transgene can function as a "selectable marker" for gain or loss of function. The use of inserted or targeted transgenes as selection tools enables positive selection without the use of an added fluorescent marker (e.g., GFP, RFP) or antibiotic selection gene. In certain cases, targeted insertion of a transgene can inactivate the target locus, such that loss of function can be monitored or selected for, e.g., inactivation of the GGTA1 locus eliminates or reduces binding of targeted cells to lectin (IB4), or inactivation of B4GalNT2 eliminates or reduces binding of targeted cells to DBA lectin, and in each case, targeted incorporation can be sorted for or enriched for cells lacking such lectin binding.

[0379] Deletions can be at least about 50 bp, more usually at least about 100 bp, and generally up to about 20 kbp, where the deletion usually includes at least a portion of the coding region that includes a portion of one or more exons or one or more introns, and may or may not include a portion of an adjacent non-coding region, particularly a 5-non-coding region (transcription regulatory region). Thus, the homologous region can extend beyond the coding region into the 5'-non-coding region or into the 3'-non-coding region. Insertions generally cannot exceed 10 kbp, usually cannot exceed 5 kbp, and generally are at least 50 bp, more usually at least 200 bp.

[0380] The region(s) of homology can include mutations, where the mutations can further inactivate the target gene when providing a frameshift or changing an important amino acid, or the mutations can correct a dysfunctional allele or the like. Usually, the mutations do not exceed about 5% of the homologous adjacent sequence or can even be slight changes such as single nucleotide changes like point mutations at the active site of an exon. When mutations in the gene are desired, the marker gene can be inserted into an intron so that it is excised from the target gene during transcription.

[0381] Various considerations, such as the size of the target locus, the availability of the sequence, the relative efficiency of double crossover events at the target locus, and the similarity between the target sequence and other sequences, can be involved in determining the degree of homology of the target DNA sequence. The targeting DNA can include DNA that is substantially homologous to the corresponding target sequence in the genome to be modified and that flanks the desired sequence modification. Substantially homologous sequences can be at least about 95%, or at least about 97% or at least about 98% or at least about 99% or between 95% and 100%, 97 - 98%, 99.0 - 99.5%, 99.6 - 99.9% or 100% identical to the corresponding target sequence (excluding the desired sequence modification). In certain embodiments, the targeting DNA and the target DNA can share a stretch of DNA that is 100% identical for at least about 75, 150 or 500 base pairs. Thus, the targeting DNA can be derived from cells that are closely related to the cell line being targeted; or the targeting DNA can be derived from cells of the same cell line or animal as the cell being targeted.

[0382] Constructs can be prepared according to methods known in the art, various fragments can be joined together, introduced into a suitable vector, cloned, analyzed, and then further manipulated until the desired construct is achieved. Various modifications can be made to the sequence to enable restriction analysis, excision, identification of probes, etc. Silent mutations can be introduced as desired. At various stages, restriction analysis, sequencing, amplification by polymerase chain reaction, primer repair, in vitro mutagenesis, etc. can be used.

[0383] The construct can be prepared using a bacterial vector containing an origin recognizable by a prokaryotic replication system, e.g., E. coli, and at each stage, the construct can be cloned and analyzed. A marker the same as or different from the marker used for insertion can be used, which can be removed prior to introduction into the target cells. Once the vector containing the construct is completed, it can be further manipulated, e.g., by deletion of bacterial sequences, linearization, introduction of short deletions in homologous sequences. After the final manipulation, the construct can be introduced into cells.

[0384] Techniques that can be used to enable entry of a DNA or RNA construct into a host cell include calcium phosphate / DNA co-precipitation, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, lipofection, infection, particle bombardment, or any other technique known to those skilled in the art. The DNA or RNA can be single-stranded or double-stranded, linear or circular, relaxed or supercoiled DNA. For various techniques for transfecting mammalian cells, see, e.g., Keown et al., Methods in Enzymology, Vol. 185, pp. 527-537 (1990).

[0385] The following vectors are provided by way of example. Bacteria: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR54O, pRIT5 (Pharmacia). Eukaryotes: pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmiacia). Also, any other plasmid and vector may be used as long as it is replicable and viable in the host. Vectors known in the art and commercially available vectors (and their variants or derivatives) may be engineered to contain one or more recombination sites for use in the methods of the present invention according to the present invention. Such vectors can be obtained, for example, from Vector Laboratories Inc., Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen and Research Genetics.Other vectors for the purpose include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110 and pKK232-8 (Pharmacia, Inc.), p3’SS, pXT1, pSG5, pPbac, pMbac, pMC1neo and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B and C, pVL1392, pBlueBaclll, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4 and pEBVHis (Invitrogen, Corp.), and their variants or derivatives.

[0386] Other vectors include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), P1 (Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vector, PhageScript vector, BlueScript vector, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSY--SPORT1 (Invitrogen) and their variants or derivatives. Viral vectors such as lentiviral vectors can also be used (see, for example, WO03 / 059923; Tiscornia et al. PNAS 10 0 vol: 1844-1848 (2003)).

[0387] Additional vectors for the purpose include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1 / His, pcDNA3.1(-) / Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO81S, pPICZ, pPICZA, pPICZB, pPICZC, pGAPZA, pGAPZB, pGAPZC, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1 / Amp, pcDNA3.1, pcDNA3.1 / Zeo, pSe, SV2, pRc / CMV2, pRc / RSV, pREP4, pREP7, pREP8, pREP9, pREP10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni and pCRBac from Invitrogen; λExCell, λgt11, pTrc99A, pKK223-3, pGEX-1λT, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO and pUC4K from Pharmacia; pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4-abc(+), pOCUS-2, pTAg, pET-32L1C, pET-30LIC, pBAC-2 cp LIC, pBACgus-2 cp LIC, pT7Blue-2 LIC, pT7Blue-2, λSCREEN-1, λBlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11 from Novagen abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b - pET-17xb, pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3 cp, pBACgus-2 cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg and Selecta Vecta-Gpt; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP--N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic, pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMV, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1 / 2 / 3, pYEX-S1, pBacPAK-His, pBacPAK8 / 9, pAcUW31, BacPAK6, pTrip1Ex, 2λgt10, λgt11, pWE15 and λTrip1Ex;Included are Stratagene's λZAP II, pBK-CMV, pBK-RSV, pBluescript II KS+ / - , pBluescript II SK+ / - , pAD-GAL4, pBD-GAL4 Cam, pSurfscript, λFIX II, λDASH, λEMBL3, λEMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS+ / - , pBC KS+ / - , pBC SK+ / - , Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI / MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415 and pRS416.;

[0388] Further vectors include, for example, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and their variants or derivatives.

[0389] In an exemplary embodiment, the vector is a bicistronic vector. The bicistronic vector includes one promoter and two transgenes. In a particular embodiment, the bicistronic vector includes one promoter and two transgenes linked by a 2A sequence. This embodiment enables the simultaneous expression of multiple functional transgenes from a single transcript. More specifically, this embodiment utilizes a short (18 - 24 aa) cleavage peptide "2A", which enables the co-expression of linked open reading frames so as to express functional transgenes from a single transcript 2A vector system.

[0390] In an exemplary embodiment, the vector is a multicistronic vector (MCV). In one embodiment, the MCV includes one promoter and at least four transgenes. In a particular embodiment, the MCV includes four transgenes linked by a 2A peptide sequence under the control of at least two promoters. This embodiment enables the simultaneous expression of multiple functional transgenes from a single transcript. More specifically, this embodiment utilizes a short (18 - 24 aa) cleavage peptide "2A", which enables the co-expression of linked open reading frames so as to express functional transgenes from a single transcript 2A vector system.

[0391] In an exemplary embodiment, the vector is a 2A-peptide MCV vector that includes at least two bicistronic units, and each bicistronic unit includes two transgenes. In a particular embodiment, one bicistronic unit is controlled by a constitutive or ubiquitous promoter (e.g., CAG), and the second bicistronic unit is controlled by an endothelial or other tissue-specific or inducible promoter system. In certain particular embodiments, only at least four transgenes are inserted at a single locus, but each is controlled by its own promoter or by a total of at least two promoters per single locus insertion.

[0392] In an exemplary embodiment, the vector is a 4-gene MCV comprising at least two anticoagulation factors, more particularly at least three anticoagulation factors.

[0393] In an exemplary embodiment, the vector is a 4-gene MCV vector comprising at least two anticoagulation factors and one complement inhibitor, more particularly three anticoagulation factors and one complement inhibitor.

[0394] In an exemplary embodiment, the vector is a 4-gene MCV vector comprising two anticoagulation factors, one complement inhibitor and one immunosuppressive factor.

[0395] Promoter The vector construct used to produce the animals of the present invention may include regulatory sequences, including but not limited to promoter-enhancer sequences operably linked to the sequence, "2A" peptide technology and docking vectors. A number of suitable vectors and promoters are known to those skilled in the art and are commercially available.

[0396] In a specific embodiment, the present invention provides animals, tissues and cells that express at least one transgene (in combination with at least one transgene under the control of a second same or different promoter) in endothelial cells, more particularly at least two, at least three or at least four transgenes in endothelial cells. To target expression to a particular tissue, the animal is developed using a vector comprising a promoter specific for endothelial cell expression. In certain embodiments, expression is controlled primarily by promoter activity in the endothelium.

[0397] In one embodiment, the nucleic acid construct comprises a regulatory sequence operably linked to an expressed transgene sequence. In one embodiment, this regulatory sequence can be a promoter sequence. In one embodiment, this promoter can be a regulatable promoter. In such a system, a drug can be used, for example, to regulate whether a peptide is expressed in an animal, tissue or organ. For example, expression can be prevented while the organ or tissue is part of a pig, but expression is induced when the pig is transplanted into a human over a certain period to overcome the cellular immune response. Further, the level of expression can be controlled by a regulatable promoter system to ensure that immunosuppression of the recipient's immune system does not occur. The regulatable promoter system can be selected from, but is not limited to, the following gene systems: the metallothionein promoter inducible by a metal such as copper (see Lichtlen and Schaffner, Swiss Med. Wkly., 2001, 131(45 - 46):647 - 52); the tetracycline-regulated system (see Imhof et al., J Gene Med., 2000, 2(2):107 - 16); the ecdysone-regulated system (see Saez et al., Proc Natl Acad Sci USA., 2000, 97(26):14512 - 7); the cytochrome P450 inducible promoter, for example, the CYP1A1 promoter (see Fujii-Kuriyama et al., FASEB J., 1992, 6(2):706 - 10); the mif Sirin- and Park-inducible systems (see Sirin and Park, Gene., 2003, 323:67-77); coumarin-activated systems (see Zhao et al., Hum Gene Ther., 2003, 14(17):1619-29); macrolide-inducible systems (responsive to macrolide antibiotics such as rapamycin, erythromycin, clarithromycin, and roxitiromycin) (see Weber et al., Nat Biotechnol., 2002, 20(9):901-7; Wang et al., Mol Ther., 2003, 7(6):790-800); ethanol-induced systems (see Garoosi et al., J Exp Bot., 2005, 56(416):1635-42; Roberts et al., Plant Physiol., 2005, 138(3):1259-67); streptogramin-inducible systems (see Fussenegger et al., Nat Biotechnol., 2000, 18(11 ):1203-8); electrophile-inducible systems (see Zhu and Fahl, Biochem Biophys Res Commun., 2001, 289(1):212-9). ; nicotine-inducible systems (see Malphettes et al., Nucleic Acids Res., 2005, 33(12):e107), immune-inducible promoters, cytokine-responsive promoters (e.g., promoters that are induced by IFN-gamma, TNF-alpha, IL-1, IL-6, or TGF-beta (or other secondary pathways) and thus can be switched on or upregulated in association with or in response to an immune or inflammatory response).

[0398] In certain embodiments, the dicistronic vector comprises two transgenes and one promoter that is primarily active in endothelial cells or one constitutive promoter that ubiquitously expresses the transgenes in all organs, tissues, and cells. In other embodiments, at least four transgenes in a multicistronic vector (MCV) are under the control of at least two promoters. These promoters can be exogenous, native, or a combination of both exogenous and native.

[0399] In certain embodiments, the dicistronic vector comprises two transgenes and one constitutive promoter that ubiquitously expresses the transgenes in all organs, tissues, and cells. In certain embodiments, the dicistronic vector comprises two transgenes and one tissue-specific promoter that controls expression in organs, tissues, and cells.

[0400] In an exemplary embodiment, the vector is a 4-gene MCV comprising at least two anticoagulation factors under the control of one endothelium-specific promoter.

[0401] In an exemplary embodiment, the vector is a 4-gene MCV comprising at least one complement inhibitor transgene under the control of one constitutive promoter and at least one anticoagulation factor transgene under the control of an endothelium cell-specific promoter.

[0402] In an exemplary embodiment, the vector is a 4-gene MCV comprising at least one complement inhibitor transgene under the control of one constitutive promoter and at least one anticoagulation factor gene under the control of a second constitutive promoter.

[0403] In an exemplary embodiment, the vector is a 4-gene MCV vector comprising one anticoagulation factor transgene and one immunosuppressive factor transgene under the control of one endothelium cell promoter.

[0404] In an exemplary embodiment, the vector is a 2-gene MCV vector that includes a total of two genes under the control of at least two separate promoters; or in a selected embodiment, a vector having a plurality of transgenes, each having its own promoter and all integrated into a single locus.

[0405] In other embodiments, enhancer elements are used in nucleic acid constructs to promote increased expression of transgenes in a tissue-specific manner. An enhancer is an outer element that significantly alters the efficiency of gene transcription (Molecular Biology of the Gene , 4th Edition, pp. 708-710, Benjamin Cummings Publishing Company, Menlo Park, Calif. (Copyright) 1987). In certain embodiments, the pdx-1 enhancer (also known as IPF-1, STF-1, and IDX1; Gerrish K et al., Mol. Endocrinol., 2004, 18(3):533; Ohlsson et al., EMBO J., November 1993, 1 2(11):4251-9; Leonard et al., Mol. Endocrinol., 1993, 7(10):1275-83; Miller et al., EMBO J., 1994, 13(5):1145-56; Serup et al., Proc Natl Acad Sci USA., 1996, 93(17):9015-20; Melloul et al., Diabetes., 2002, 51 Suppl 3:S320-5; Glick et al., J Biol Chem., 2000, 275(3):2199-204; GenBank AF334615.)) is used in combination with the ins2 promoter for pancreatic-specific expression of the transgene(s). In certain embodiments, the animal expresses the transgene under the control of a promoter combined with an enhancer element. In certain embodiments, the animal contains an endothelium-specific promoter, such as the porcine ICAM-2 or murine Tie-2 promoter, and further contains an enhancer element (e.g., murine Tie-2 enhancer or CMV enhancer). In other embodiments, the promoter can be a ubiquitous promoter element that further contains an enhancer element. In certain elements, the ubiquitous promoter is CAG (CMV enhancer, chicken beta-actin promoter, rabbit beta-globin intron) (Tie2-CAG) used in combination with an endothelium-specific Tie-2 enhancer element. For Tie2-CAG, the transgene(s) is predicted to be expressed in both a constitutive or ubiquitous manner, but is predicted to be expressed at a higher level in endothelial cells compared to other body cells. In some embodiments, the promoter is used in combination with an enhancer element that is a non-coding region or intron region of DNA that is intrinsically related to or co-localizes with that promoter. In another specific embodiment, the enhancer element is ICAM-2 used in combination with the ICAM-2 promoter. Other ubiquitous promoters include, but are not limited to: viral promoters such as CMV and SV40, as well as chicken beta-actin and gamma-actin promoters, GAPDH promoter, H2K, CD46 promoter, GGTA1, ubiquitin, and ROSA promoter.

[0406] (v) Selection of Genetically Modified Cells In some cases, the transgenic cells have a genetic modification that is the result of the insertion or incorporation of a targeted transgene into the cell genome (i.e., via homologous recombination). In some cases, the transgenic cells have a genetic modification that is the result of non-targeted (random) incorporation into the cell genome. These cells can be grown in a suitably selected medium to identify the cells that provide the appropriate incorporation. Then, the cells that exhibit the desired phenotype can be further analyzed by restriction analysis, electrophoresis, Southern analysis, polymerase chain reaction, or another technique known in the art. By identifying a fragment that shows appropriate insertion at the target gene site (or in the case of non-targeted applications, where the random incorporation technique produced the desired result), cells in which homologous recombination (or the desired non-targeted incorporation event) has occurred to inactivate or otherwise modify the target gene can be identified.

[0407] The presence of a selectable marker gene or other positive selection agent or transgene demonstrates the incorporation of the target construct into the host genome. Then, the cells that exhibit the desired phenotype can be further analyzed by restriction digestion analysis, electrophoresis, Southern analysis, polymerase chain reaction, etc. to demonstrate whether homologous recombination or non-homologous recombination has occurred. This can be determined by using a probe for the insert and then sequencing the 5' and 3' regions adjacent to this insert for the presence of genes extending beyond the adjacent regions of the construct, or by identifying the presence of such deletions if deletions are introduced. Primers complementary to sequences within the construct, and primers complementary to sequences outside the construct at the target gene locus, can also be used. In this method, when homologous recombination occurs, only the DNA duplex having both primers present in the complementary strands can be obtained. For example, the occurrence of homologous recombination is supported by demonstrating the presence of primer sequences or sequences of the predicted size.

[0408] The polymerase chain reaction used to screen for identical recombination events was described by Kim and Smithies, (1988) Nucleic Acids Res. 16:8887-8903; and by Joyner et al. (1989) Nature 338:153-156.

[0409] Cell lines obtained from the first round of targeting (or from non-targeted (random) integration into the genome) are likely to be heterozygous for the incorporated alleles. Homozygosity in which both alleles are modified can be achieved in several ways. One approach is to grow several cells in which one copy has been modified and then subject these cells to another round of targeting (or non-targeted (random) integration) using different selectable markers. Alternatively, homozygotes can be obtained by mating animals that are heterozygous for the modified allele. In some situations, it may be desirable to have two different modified alleles. This can be achieved by successive rounds of gene targeting (or random integration) or by mating heterozygotes each having one of the desired modified alleles. Genome editing events by efficient targeted double-strand breaks enable frequent biallelic gene targeting events, such that in a single transfection (or embryo or zygote targeting strategy), homozygous knockout or knock-in events can be achieved at high frequency. Such gene editing-enhanced (e.g., Crispr-CAS9 nuclease) gene targeting or homology-dependent repair events include both monoallelic or heterozygous knockouts and biallelic or homozygous knockouts (via small nucleotide insertions, deletions, substitutions, i.e., INDELs), as well as also gene insertions including both monoallelic and biallelic insertions / knock-ins of a single transgene, multiple transgene strings (transgenes under their own promoters or bicistronic or multicistronic strings), or multicistronic vectors (including a 4-transgene multicistronic vector under the control of at least 2 promoters, said promoters being constitutive or tissue-specific, e.g., CAG and Icam-2).Alternatively, it can be predicted that cells (via transfection or infection) or zygotes (simultaneously via microinjection) having a combination of a basal genotype (i.e., GGTA1 knockout or GGTA1 / CD46) can be efficiently produced through the use of a plurality of gene editing nucleases (e.g., Crispr / Cas9), where one gene modification is a knock-in (e.g., in GGTA1) or random insertion of a 4-gene MCV (under the control of at least two promoters), and simultaneously, a nuclease-mediated INDEL at another locus (e.g., at one or two alleles in GGTA1 or CMAH or B4GalNT2), or in a preferred embodiment, a targeted insertion of a multi-transgene vector (bicistron or 4-gene MCV) at two different loci (landing pads, safe harbors, or GGTA1, B4GalNT2, CMAH, ROSA26, AAVS1 or other predetermined loci, including natural or modified natural loci), e.g., a targeted insertion of a 4-gene MCV in GGTA1 accompanied by a targeted homologous recombination (or gene editing enhanced) insertion of a bicistron or 4-gene MCV at a second locus (e.g., CMAH or B4GalNT2). In certain embodiments, selection techniques are used to obtain homozygous knockout cells from heterozygous cells by exposure to very high levels of a selective agent. Such selection is obtained, for example, by the use of an antibiotic such as geneticin (G418).

[0410] Subsequently, cells that have been transfected or received an appropriate vector by other means can be selected or identified through genotypic or phenotypic analysis. In one embodiment, the cells are grown in an appropriately selected medium to identify cells containing the transfected and incorporated vector. The presence of a selectable marker gene indicates the presence of the transgene construct in the transfected cells. Cells that exhibit the desired phenotype can then be further analyzed by restriction analysis, electrophoresis, Southern analysis, polymerase chain reaction, etc. to analyze the DNA in order to verify the integration of the transgene(s) into the genome of the host cell. Primers complementary to the transgene sequence(s) can also be used. The polymerase chain reaction, which is used to screen for homologous recombination and random integration events, is known in the art. See, for example, Kim and Smithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al., Nature 338:153-156, 1989. A specific combination of a mutant polyoma enhancer and a thymidine kinase promoter for driving the neomycin gene has been shown to be active in both embryonic stem cells and EC cells by Thomas and Capecchi, supra, 1987; Nicholas and Berg (1983), Teratocarcinoma Stem Cell, Siver, Martin and Strickland eds. (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (469-497); and Linney and Donerly, Cell 35:693-699, 1983.

[0411] Cells that have undergone the same recombination can be identified by several methods. In one embodiment, the selection method can detect the absence of an immune response against the cells, for example, by a human anti-gal antibody. In a preferred embodiment, this selection method can utilize an inserted or targeted transgene as a selection tool that enables positive selection without the use of an added fluorescent marker (e.g., GFP, RFP) or an antibiotic selection gene. In certain cases, targeted insertion of a transgene can produce a cell surface protein, whereby appropriately transgene-specific fluorescently marked cells can be sorted for positive expression of the desired transgene. Alternatively, the target locus can be inactivated, such that loss of function can be monitored or selected for, e.g., inactivation of the GGTA1 locus eliminates or reduces binding of targeted cells to lectin (IB4), or inactivation of B4GalNT2 eliminates or reduces binding of targeted cells to DBA lectin, and in each case, targeted incorporation can result in sorting or enrichment for cells lacking such lectin binding. In each case, expression of the transgene on the cell surface enables selection of cells for further analysis.

[0412] In other embodiments, this selection method may include assessing the level of blood clotting in human blood when exposed to cells or tissues. Selection via antibiotic resistance has been most commonly used for screening. This method can detect the presence of a resistance gene on the targeting vector, but does not directly indicate whether the incorporation is a targeted recombination event or a random incorporation. Alternatively, the marker can be a fluorescent marker gene, e.g., GFP or RFP, or a gene detectable on the cell surface via cell sorting or FACS analysis. Certain technologies, e.g., polyA and promoter trap technologies, increase the probability of a targeted event, but again do not give direct evidence that the desired phenotype has been achieved. Additionally, negative selection can be used to select for targeted incorporation; in these cases, genes for factors lethal to the cell (e.g., Tk or diphtheria A toxin) are inserted in such a way that only the targeted event allows the cell to avoid death. The cells selected by these methods can then be assayed for gene disruption, vector integration, and ultimately gene depletion. In these cases, the selection is based on detection of targeting vector integration and not in the altered phenotype, so simple targeted knockouts, gene rearrangements or truncations, or other such modifications that are not point mutations can be detected. The targeted events are inserted in such a way that only they allow the cell to avoid death. The cells selected by these methods can then be assayed for gene disruption, vector integration, and ultimately gene depletion. In these cases, the selection is based on detection of targeting vector integration and not in the altered phenotype, so simple targeted knockouts, gene rearrangements or truncations, or other such modifications that are not point mutations can be detected.

[0413] Characterization can be further achieved by techniques including, but not limited to, PCR analysis, Southern blot analysis, Northern blot analysis, specific lectin binding assays, and / or sequencing analysis. Phenotypic characterization can also be achieved, including binding of anti-mouse antibodies in various assays, including tests for transcription of RNA in cells in immunofluorescence, immunocytochemistry, ELISA assays, flow cytometry, Western blotting, RT-PCR, etc. Genotype can be determined by Southern analysis and PCR. Gene expression is monitored by flow cytometry of PBMC and endothelial cells, as well as by immunohistochemistry, Q-PCR (quantitative polymerase chain reaction), and Western blot analysis in cells and organs. Bioactivity assays specific for the transgene quantify and characterize complement inhibition, platelet aggregation, activated protein C formation, ATPase activity, factor Xa cleavage, mixed lymphocyte reaction (MLR), and apoptosis.

[0414] In other embodiments, the GTKO animals or cells contain further genetic modifications. The genetic modifications can include more than homologous targeting, but can also include random integration of exogenous genes, co-integration of groups or strings of genes at a single locus, mutation, deletion, and insertion of any kind of gene. Further genetic modifications can be made by further genetically modifying cells obtained from the transgenic cells and animals described herein, or by mating the animals described herein with further genetically modified animals. Such animals can be modified to eliminate the expression of at least one allele of the alpha GT gene, the CMP-Neu5Ac hydroxylase gene (see, e.g., U.S. Patent No. 7,368,284), the iGb3 synthase gene (see, e.g., U.S. Patent Application Publication No. 2005 / 0155095), and / or beta1,4 N-acetylgalactosaminyltransferase (β4GalNT2; see, e.g., Estrada JL et al., Xenotransplantation 22:194-202

[2015] ), the Forssman synthase gene (see, e.g., U.S. Patent Application Publication No. 2006 / 0068479).

[0415] In further embodiments, the animals described herein can also include genetic modifications such that they express a transgene of interest, more specifically a human transgene, from the group consisting of immunomodulatory factors, anticoagulation factors, and cytoprotective transgenes. In preferred embodiments, in addition to multi-transgene integration (targeted or random, but exceeding at least four genes, and such at least four genes being controlled by at least two promoters), gene modifications of the porcine vWF locus can be achieved that include knockout (loss of function), INDEL, and co-knockout of the porcine vWF sequence in the genome, or targeted knock-in and replacement of some or all of the defined porcine vWF exons (e.g., exons 22-28) by their human exon 22-28 counterparts from the human vWF gene sequence.

[0416] To achieve these further genetic modifications, in one embodiment, the cells can be modified to include multiple genetic modifications. In other embodiments, animals can be mated together to achieve multiple genetic modifications. In a specific embodiment, an animal such as a pig produced according to the processes, sequences and / or constructs described in the present invention can be mated with an animal such as a pig lacking the expression of alphaGal (e.g., as described in WO04 / 028243).

[0417] In another embodiment, the expression of further genes responsible for xenograft rejection can be eliminated or reduced. Such genes include, but are not limited to, the CMP-NEUAc hydroxylase gene (CMAH), beta-4GalNT2, isoGloboside 3 (iGb3) synthase gene and the Forssman synthase gene.

[0418] Furthermore, genes or cDNAs encoding complement-related proteins responsible for suppressing complement-mediated lysis can also be expressed in the animals and tissues of the present invention. Such genes include, but are not limited to, CD59, DAF (CD55) and CD46 (see, for example, WO99 / 53042; Chen et al. Xenotransplantation, Vol. 6, No. 3, pages 194 - August 1999, describing pigs expressing the CD59 / DAF transgene; Costa C et al. Xenotransplantation. January 2002; 9(1):45 - 57, describing transgenic pigs expressing human CD59 and H-transferrin; Zhao L et al., Diamond L E et al. Transplantation. January 15, 2001; 71(1):132 - 42, describing human CD46 transgenic pigs). See also Costa C et al. Xenotransplantation. January 2002; 9(1):45 - 57, describing transgenic pigs expressing human CD59 and H-transferrin; Zhao L et al., Diamond L E et al. Transplantation. January 15, 2001; 71(1):132 - 42, describing human CD46 transgenic pigs).

[0419] Further modifications include, as described in WO00 / 31126, entitled "Suppression of xenograft rejection by down regulation of a cell adhesion molecules" Compounds such as antibodies that downregulate the expression of cell adhesion molecules by naive cells, and CTLA-4 in soluble form from xenogeneic donor organisms as described in WO99 / 57266, title "Immunosuppression by blocking T cell co-stimulation signal 2 (B7 / CD28 interaction)", etc., may be included in the administration of such compounds to organ recipients to prevent co-stimulation by signal 2. -4

[0420] (vi) Nuclear transfer The genetically modified or transgenic animals described herein, such as ungulates or pigs, can be produced using any suitable technique known in the art. These techniques include, but are not limited to, microinjection (e.g., of pronuclei and / or cytoplasm), electroporation of oocytes or zygotes, and / or somatic cell nuclear transfer (SCNT).

[0421] Any additional techniques known in the art can be used to introduce the transgene, or multiple transgenes or MCV vector(s) into the animal. Such techniques include pronuclear microinjection (see, e.g., Hoppe, P.C. and Wagner, T.E., 1989, U.S. Patent No. 4,873,191); cytoplasmic microinjection (see, e.g., Whitworth et al., 2014); retrovirus-mediated gene transfer into the germ line (see, e.g., Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82: 6148-6152); gene targeting in embryonic stem cells (see, e.g., Thompson et al., 1989, Cell 56: 313-321; Wheeler, M.B., 1994, WO94 / 26884); electroporation of embryos (see, e.g., Lo, 1983, Mol Cell. Biol. 3: 1803-1 804); etc. See page 814); transfection; transduction; retroviral infection; adenoviral infection; adenovirus-related infection; liposome-mediated gene transfer; naked DNA transfer; and sperm-mediated gene transfer (e.g., Lavitrano et al., 1989, Cell 57 : see pages 717-723), etc., but not limited thereto. For an overview of such techniques, see, for example, Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115: see pages 171-229. In certain embodiments, expression of CTLA4 and / or CTLA4-Ig fusion genes in ungulates can be achieved via these techniques.

[0422] In one embodiment, microinjection of a construct encoding a transgene can be used to produce a transgenic animal. In one embodiment, a nucleic acid construct or vector can be microinjected into the pronucleus of a zygote. In one embodiment, the construct or vector can be injected into the male pronucleus of a zygote. In another embodiment, the construct or vector can be injected into the female pronucleus of a zygote. In a further embodiment, a construct or vector, messenger RNA (mRNA) encoding CRISPR(s), Cas9 and guide RNA (single guide RNA) can be injected into the cytoplasm of a fertilized oocyte to achieve gene knockout or gene inactivation (insertion, deletion, substitution) resulting from repair errors after treatment with such gene editing nucleases, or to achieve targeted knock-in of a transgene(s) or multi-gene vector in such zygotes to obtain stable transmission of genetic modification (reference, Whitworth 2014?). In another embodiment, nuclear transfer is performed on an existing transgenic somatic It can be initiated in cells, and after embryo reconstruction and fusion, a gene editing nuclease (e.g., Crispr / Cas9) can be injected into the cytoplasm of the reconstructed nuclear transfer embryo, with or without a transgene vector, or a multi-gene vector or MCV, such that gene editing events occur in the diploid embryo and in the subsequent transgenic pig after embryo transfer.

[0423] Microinjection of a transgene construct or vector can include the following steps: su...

Claims

[Claim 1] A transgenic pig comprising at least four introduced genes, (i) the at least four transgenes are integrated and expressed at a single locus under the control of at least two promoters; (ii) the single locus is a native or modified locus selected from the group consisting of CMAH, β4GalNT2, and GGTA1; (iii) the transgenic pig lacks expression of alpha 1,3 galactosyltransferase; and (iv) two of the at least four transgenes are expressed as a first polycistron controlled by a first promoter and two of the four transgenes are expressed as a second polycistron controlled by a second promoter different from the first promoter; and (a) the first polycistron comprises TBM and CD39; the second polycistron comprises EPCR and DAF (CD55); TFPI and CD47; EPCR and CD47; EPCR and HO-1; CD47 and HO-1; A20 and CD47; CIITAKD and HO-1; CTLA4Ig and TFPI; CIITAKD and A20; CIITAKD and TFPI; EPCR and TFPI; or CIITA and TFPI; (b) the first polycistron comprises EPCR and DAF (CD55); the second polycistron comprises TBM and CD39; TFPI and CD47; TBM and CD47; TBM and TFPI; TBM and HO-1; CD47 and HO-1; A20 and CD47; CIITAKD and HO-1; CTLA4Ig and TFPI; CIITAKD and A20; TBM and A20; CIITAKD and TFPI; or CIITA and TFPI; (c) the first polycistron comprises TFPI and CD47; the second polycistron comprises TBM and CD39; EPCR and DAF (CD55); EPCR and HO-1; TBM and HO-1; TBM and EPCR; CIITAKD and HO-1; CIITAKD and A20; or TBM and A20; (d) the first polycistron comprises TBM and CD47; the second polycistron comprises EPCR and DAF (CD55); EPCR and HO-1; CIITAKD and HO-1; CTLA4Ig and TFPI; CIITAKD and A20; CIITAKD and TFPI; EPCR and TFPI; or CIITA and TFPI; (e) the first polycistron comprises EPCR and CD47; the second polycistron comprises TBM and CD39; TBM and TFPI; TBM and HO-1; CIITAKD and HO-1; CTLA4Ig and TFPI; CIITAKD and A20; TBM and A20; CIITAKD and TFPI; or CIITA and TFPI; (f) the first polycistron comprises TBM and TFPI; the second polycistron comprises EPCR and DAF (CD55); EPCR and CD47; EPCR and HO-1; CD47 and HO-1; A20 and CD47; CIITAKD and HO-1; or CIITAKD and A20; (g) the first polycistron comprises EPCR and HO-1; the second polycistron comprises TBM and CD39; TFPI and CD47; TBM and CD47; TBM and TFPI; TFPI and CD47; A20 and CD47; CTLA4Ig and TFPI; CIITAKD and A20; TBM and A20; CIITAKD and TFPI; or CIITA and TFPI; (h) the first polycistron comprises TBM and HO-1; the second polycistron comprises EPCR and DAF (CD55); TFPI and CD47; EPCR and CD47; TFPI and CD47; A20 and CD47; CTLA4Ig and TFPI; CIITAKD and A20; CIITAKD and TFPI; EPCR and TFPI; or CIITA and TFPI; (i) the first polycistron comprises CD47 and HO-1; the second polycistron comprises TBM and CD39; EPCR and DAF (CD55); TBM and TFPI; TBM and EPCR; CTLA4Ig and TFPI; CIITAKD and A20; TBM and A20; CIITAKD and TFPI; EPCR and TFPI; or CIITA and TFPI; (j) the first polycistron comprises TBM and EPCR; the second polycistron comprises TFPI and CD47; CD47 and HO-1; TFPI and CD47; A20 and CD47; CIITAKD and HO-1; CTLA4Ig and TFPI; CIITAKD and A20; CIITAKD and TFPI; or CIITA and TFPI; (k) the first polycistron comprises A20 and CD47; the second polycistron comprises TBM and CD39; EPCR and DAF (CD55); TBM and TFPI; EPCR and HO-1; TBM and HO-1; TBM and EPCR; CIITAKD and HO-1; CTLA4Ig and TFPI; CIITAKD and TFPI; EPCR and TFPI; or CIITA and TFPI; (l) the first polycistron comprises CIITAKD and HO-1; the second polycistron comprises TBM and CD39; EPCR and DAF (CD55); TFPI and CD47; TBM and CD47; EPCR and CD47; TBM and TFPI; TFPI and CD47; TBM and EPCR; A20 and CD47; CTLA4Ig and TFPI; TBM and A20; EPCR and TFPI; or CIITA and TFPI; (m) the first polycistron comprises CTLA4Ig and TFPI; the second polycistron comprises TBM and CD39; EPCR and DAF (CD55); TBM and CD47; EPCR and CD47; EPCR and HO-1; TBM and HO-1; CD47 and HO-1; TBM and EPCR; A20 and CD47; CIITAKD and HO-1; CIITAKD and A20; or TBM and A20; (n) the first polycistron comprises CIITAKD and A20; the second polycistron comprises TBM and CD39; EPCR and DAF (CD55); TFPI and CD47; TBM and CD47; EPCR and CD47; TBM and TFPI; EPCR and HO-1; TBM and HO-1; CD47 and HO-1; TFPI and CD47; TBM and EPCR; CTLA4Ig and TFPI; EPCR and TFPI; or CIITA and TFPI; (o) the first polycistron comprises TBM and A20; the second polycistron comprises EPCR and DAF (CD55); TFPI and CD47; EPCR and CD47; EPCR and HO-1; CD47 and HO-1; TFPI and CD47; CIITAKD and HO-1; CTLA4Ig and TFPI; CIITAKD and TFPI; EPCR and TFPI; or CIITA and TFPI; (p) the first polycistron comprises CIITAKD and TFPI; the second polycistron comprises TBM and CD39; EPCR and DAF (CD55); TBM and CD47; EPCR and CD47; EPCR and HO-1; TBM and HO-1; CD47 and HO-1; TBM and EPCR; A20 and CD47; CTLA4Ig and TFPI; or TBM and A20; (q) the first polycistron comprises EPCR and TFPI; the second polycistron comprises TBM and CD39; TBM and CD47; EPCR and CD47; TBM and HO-1; CD47 and HO-1; A20 and CD47; CIITAKD and HO-1; CTLA4Ig and TFPI; CIITAKD and A20; or TBM and A20; or (r) the first polycistron comprises CIITA and TFPI; the second polycistron comprises TBM and CD39; EPCR and DAF (CD55); TBM and CD47; EPCR and CD47; EPCR and HO-1; TBM and HO-1; CD47 and HO-1; TBM and EPCR; A20 and CD47; CIITAKD and HO-1; CIITAKD and A20; or TBM and A20. Transgenic pigs.

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